Ultra-long acting insulin-Fc fusion proteins and methods of use

ABSTRACT

The present disclosure provides recombinantly manufactured ultra-long acting insulin-Fc fusion proteins for use in treating canine and feline diabetes. The insulin-Fc fusion proteins comprise an insulin polypeptide linked via a peptide linker to an Fc-fragment of canine or feline origin. Based on the results obtained, creating a treatment that is amenable to low cost manufacturing, exhibits sufficient in vivo bioactivity, displays extended duration of bioactivity, does not induce anti-drug antibodies, and substantially retains is potency over multiple administrations, requires a non-obvious combination of insulin polypeptide, peptide linkers, and species-specific Fc fragment, in addition to selective mutations on one or more of these components. Exemplary ultra-long acting insulin-Fc fusion proteins, polynucleotides encoding these insulin-Fc fusion proteins, and pharmaceutical formulations of exemplary insulin-Fc fusion proteins are provided, in addition to methods of use and preparation.

PRIORITY AND RELATED APPLICATIONS

The present application is related to, claims the priority benefit of,and is a U.S. continuation patent application of, U.S. patentapplication Ser. No. 16/775,979, filed Jan. 29, 2020, which is relatedto, claims the priority benefit of, and is a U.S. bypass continuationof, PCT Patent Application Serial No. PCT/US2019/040010, filed Jun. 28,2019, which is related to, and claims the priority benefit of, U.S.Provisional Patent Application Ser. No. 62/837,188, filed Apr. 22, 2019,U.S. Provisional Patent Application Ser. No. 62/827,809, filed Apr. 1,2019, U.S. Provisional Patent Application Ser. No. 62/824,176, filedMar. 26, 2019, U.S. Provisional Patent Application Ser. No. 62/781,378,filed Dec. 18, 2018, U.S. Provisional Patent Application Ser. No.62/781,368, filed Dec. 18, 2018, U.S. Provisional Patent ApplicationSer. No. 62/774,682, filed Dec. 3, 2018, U.S. Provisional PatentApplication Ser. No. 62/743,358, filed Oct. 9, 2018, U.S. ProvisionalPatent Application Ser. No. 62/740,735, filed Oct. 3, 2018, U.S.Provisional Patent Application Ser. No. 62/719,347, filed Aug. 17, 2018,U.S. Provisional Patent Application Ser. No. 62/702,167, filed Jul. 23,2018, U.S. Provisional Patent Application Ser. No. 62/698,648, filedJul. 16, 2018, U.S. Provisional Patent Application Ser. No. 62/696,645,filed Jul. 11, 2018, U.S. Provisional Patent Application Ser. No.62/693,814, filed Jul. 3, 2018, U.S. Provisional Patent Application Ser.No. 62/692,507, filed Jun. 29, 2018, and U.S. Provisional PatentApplication Ser. No. 62/692,498, filed Jun. 29, 2018. The contents ofeach of the aforementioned patent applications are hereby incorporatedherein by reference in their entirety.

TECHNICAL FIELD

The present technology relates to compositions of insulin-Fc fusionproteins and their use to treat diabetes in companion animals, e.g.,dogs or cats.

BACKGROUND

The following description of the background of the present technology isprovided simply as an aid in understanding the present technology and isnot admitted to describe or constitute prior art to the presenttechnology.

Diabetes is a chronic condition characterized by an insulin deficiencyand/or ineffective use of insulin. Diabetics that have an absolutedeficiency of insulin are categorized as having type 1 orinsulin-dependent diabetes mellitus (IDDM). Type 1 diabetics are thoughtto have a genetic predisposition combined with immunologic destructionof the insulin-producing β-cells of the pancreas. In comparison,diabetics that can still produce some insulin but have a relativedeficiency due to insulin resistance or other dysfunction, areclassified as having type 2 or non-insulin-dependent diabetes mellitus(NIDDM). Type 2 diabetes is linked to genetic predisposition, obesity,and certain medications.

When a dog or a cat does not produce insulin or cannot use it normally,blood sugar levels elevate, resulting in hyperglycemia. Dogs generallyexhibit an atypical glycemia phenotype with strong similarities to humantype 1 diabetes. Dogs also occasionally exhibit atypical glycemia withstrong similarities to type 2 diabetes in humans. Female dogs can alsodevelop temporary insulin resistance while in heat or pregnant. In allcases, the dogs are treated with chronic insulin injection therapy. Catsgenerally exhibit an atypical glycemia phenotype with strongsimilarities to human type 2 diabetes (i.e. insulin resistance), but bythe time the disease is diagnosed by a veterinarian, it has progressedto resemble a type 1 diabetes condition (inflammatory disease inpancreas with significant loss of beta cell mass), and the cat isdependent on exogenous insulin. Some diabetic cats can be managed withdietary changes and oral medication, but the majority of diabetic catsreceive chronic insulin injection therapy to maintain adequateregulation. Left untreated, diabetes in dogs and cats can lead to weightloss, loss of appetite, vomiting, dehydration, problems with motorfunction, coma, and even death.

Approximately 0.24% of dogs and approximately 0.68% of cats in theUnited States are affected by diabetes. Current diabetes therapies fordogs and cats include the use of insulin, such as Vetsulin® for dogs(Intervet Inc., d.b.a. MERCK Animal Health, Summit, N.J.) and ProZinc®for cats (Boehringer Ingelheim Vetmedica, Duluth, Ga.) which areadministered once or twice daily. The burden of frequent injections onowners often results in a lack of treatment regimen compliance andunder-dosing, leading to poor long-term health outcomes. In fact, thecost of insulin therapy and the practicality of dosing their pets up to14 times per week leads a significant percentage of owners to selecteuthanasia for their pets as an alternative to intensive management ofdiabetes. Therefore, there is a need for cost effective and lessburdensome treatment options for this disease.

SUMMARY OF THE PRESENT TECHNOLOGY

In an aspect, the present disclosure provides a fusion proteincomprising an insulin polypeptide and an Fc fragment, wherein theinsulin polypeptide and the Fc fragment are connected by a linker, suchas a peptide linker, wherein the Fc fragment is of non-human animalorigin and comprises the following sequence:DCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 16). In some embodiments, theinsulin polypeptide of fusion protein comprises the sequenceFVNQHLCGSX1LVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCX2STCSLDQLENYCX3 (SEQ IDNO: 6), where X1 is not D, X2 is not H, and X3 is absent or N. In someembodiments, the insulin polypeptide of the fusion protein comprises thesequence FVNQHLCGSX1LVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCX2STCSLDQLENYCX3(SEQ ID NO: 6), where X1 is H, X2 is T, and X3 is absent or N. Inembodiments, the insulin polypeptide and the Fc fragment of the fusionprotein are connected by a linker, such as a peptide linker, comprisingthe sequence GGGGGQGGGGQGGGGQGGGGG (SEQ ID NO: 14).

In embodiments, the fusion protein comprises the sequenceFVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 32). Inembodiments, the fusion protein comprises the sequenceFVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCNGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 34).

In an aspect, the present disclosure provides a fusion proteincomprising an insulin polypeptide and an Fc fragment, wherein theinsulin polypeptide and the Fc fragment are connected by a linker, suchas a peptide linker, wherein the Fc fragment comprises the sequenceDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 22). In some embodiments, theinsulin polypeptide of the fusion protein comprises the sequenceFVNQHLCGSX1LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCX2STCSLDQLENYC (SEQ ID NO:10), where X1 is not D and X2 is not H. In some embodiments, the insulinpolypeptide of the fusion protein comprises the sequenceFVNQHLCGSX1LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCX2STCSLDQLENYC (SEQ ID NO:10), where X1 is H and X2 is T. In embodiments, the insulin polypeptideand the Fc fragment are connected by a linker, such as a peptide linker,comprising the sequence GGGGGQGGGGQGGGGQGGGGG (SEQ ID NO: 14).

In embodiments, the fusion protein comprises the sequenceFVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 36).

In an aspect, the present disclosure provides a fusion proteincomprising an insulin polypeptide and an Fc fragment, wherein theinsulin polypeptide and the Fc fragment are connected by a linker, suchas a peptide linker, wherein the Fc fragment is of non-human animalorigin and comprises the sequenceDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 20). In embodiments, the insulinpolypeptide of the fusion protein comprises the sequenceFVNQHLCGSX1LVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCX2STCSLDQLENYCX3 (SEQ IDNO: 6), where X1 is not D, X2 is not H, and X3 is absent. Inembodiments, the insulin polypeptide of the fusion protein comprises thesequence FVNQHLCGSX1LVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCX2STCSLDQLENYCX3(SEQ ID NO: 6), where X1 is H, X2 is T, and X3 is absent. Inembodiments, the insulin polypeptide and the Fc fragment are connectedby a linker, such as a peptide linker, comprising the following sequenceGGGGGQGGGGQGGGGQGGGGG (SEQ ID NO: 14).

In embodiments, the fusion protein comprises the sequenceFVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 38).

In an aspect, the present disclosure provides a fusion proteincomprising an insulin polypeptide and an Fc fragment, wherein theinsulin polypeptide and the Fc fragment are connected by a linker, suchas a peptide linker, wherein the Fc fragment comprises the sequenceDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFSSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 23). In embodiments, the insulinpolypeptide of the fusion protein comprises the sequenceFVNQHLCGSX1LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCX2STCSLDQLENYC (SEQ ID NO:10), where X1 is not D and X2 is not H. In embodiments, the insulinpolypeptide comprises the following sequenceFVNQHLCGSX1LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCX2STCSLDQLENYC (SEQ ID NO:10), where X1 is H and X2 is T. In embodiments, the insulin polypeptideand the Fc fragment are connected by a linker, such as a peptide linker,comprising the sequence GGGGGQGGGGQGGGGQGGGGG (SEQ ID NO: 14).

In embodiments, the fusion protein comprises the sequenceFVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFSSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 40).

In aspects, the fusion proteins described herein comprise a homodimer.In embodiments, the percentage homodimer of the fusion protein isgreater than 90%. In embodiments, the fusion proteins described hereinare made using HEK293 cells, and the resulting homodimer titer afterpurification using Protein A beads or a Protein A column is greater than50 mg/L. In embodiments, the insulin receptor IC50 for the fusionproteins described herein is less than or equal to 5000 nM. Inembodiments, the serum half-life of the fusion proteins described hereinin the blood or serum of a target animal upon administration is longerthan about 3 days. In embodiments, for the fusion proteins describedherein, the time during which there is a statistically significantdecrease in blood glucose level in a subject relative to a pre-doselevel is longer than one of 2 hours, 6 hours, 9 hours, 12 hours, 18hours, 1 day, 1.5 days, 2 days, 2.5 days, 3 days, 4 days, 5 days, 6days, 7 days, or longer.

In aspects, for the fusion proteins described herein, the NAOC after thefirst subcutaneous injection in a target animal is greater than 150%FBGL·days·kg/mg. In embodiments, for the fusion proteins describedherein, the ratio of the NAOC after the third weekly subcutaneousinjection of the fusion proteins in the target animal to the NAOC afterthe first subcutaneous injection of the fusion protein in the targetanimal is greater than 0.50.

In aspects, fusion proteins as described herein are formulated as apharmaceutical composition. In embodiments, in the pharmaceuticalcomposition the fusion protein is present at a concentration of about 3mg/mL or greater. In embodiments, the composition is suitable forsubcutaneous administration.

In an aspects, a method is described for lowering the blood glucoselevel of a target animal, the method comprising administering aphysiologically effective amount of a fusion protein as described hereinor a pharmaceutical composition thereof to the patient. In embodiments,the target animal is diagnosed with diabetes. In embodiments, the targetanimal is a dog or a cat. In some embodiments, the fusion protein isadministered subcutaneously. In some embodiments, the fusion protein isadministered daily, twice weekly, or once weekly to the target animal.In examples, the fusion protein is administered once weekly to thetarget animal at a dose between 0.025 and 0.5 mg/kg/week. In aspects, acell engineered to express a fusion protein as described here indescribed. In examples, the cell is transfected with a nucleic acidencoding the fusion protein. In examples, the cell is a HEK293 cell or aCHO cell.

In an aspect, a cDNA encoding a fusion protein as described herein isdescribed.

In embodiments, the cDNA comprises the nucleic acid sequence(SEQ ID NO: 31) atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggaactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaagtgccccgctcccgagatgctgggcggacccagcgtgttcatcttccctcccaagcccaaggacacactgctgatcgccaggaccccggaggtgacctgcgtggtggtggacctggatcccgaagaccccgaggtgcagatcagctggttcgtggatggaaagcagatgcagaccgccaagacccaaccccgggaagagcagttcaacggcacctacagggtggtgagtgtgttgcccatcggccaccaggactggctgaaggggaagcaattcacatgcaaggttaataacaaggccctgcccagccccatcgagaggaccatcagcaaggccaggggccaggcccaccagccatctgtgtacgtgctgcccccatctagggaggaactgagcaagaacacagtcagccttacttgcctgatcaaggacttcttcccaccggacatagacgtggagtggcagagtaacggccagcaggagcccgagagcaagtataggaccacaccgccccaactggacgaggacggaagctacttcctctacagcaaattgagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgcgccgtgatgcacgaggctttgcataaccactacacccaggagagcctgtc ccacagccccggatag.In embodiments, the cDNA comprises the nucleic acid sequence(SEQ ID NO: 33) atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggaactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcaacggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaagtgccccgctcccgagatgctgggcggacccagcgtgttcatcttccctcccaagcccaaggacacactgctgatcgccaggaccccggaggtgacctgcgtggtggtggacctggatcccgaagaccccgaggtgcagatcagctggttcgtggatggaaagcagatgcagaccgccaagacccaaccccgggaagagcagttcaacggcacctacagggtggtgagtgtgttgcccatcggccaccaggactggctgaaggggaagcaattcacatgcaaggttaataacaaggccctgcccagccccatcgagaggaccatcagcaaggccaggggccaggcccaccagccatctgtgtacgtgctgcccccatctagggaggaactgagcaagaacacagtcagccttacttgcctgatcaaggacttcttcccaccggacatagacgtggagtggcagagtaacggccagcaggagcccgagagcaagtataggaccacaccgccccaactggacgaggacggaagctacttcctctacagcaaattgagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgcgccgtgatgcacgaggctttgcataaccactacacccaggagagcct gtcccacagccccggatag.In embodiments, the cDNA comprises the nucleic acid sequence(SEQ ID NO: 35) atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggcactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaagtgccccgctcccgagatgctgggcggacccagcgtgttcatcttccctcccaagcccaaggacacactgctgatcgccaggaccccggaggtgacctgcgtggtggtggacctggatcccgaagaccccgaggtgcagatcagctggttcgtggatggaaagcagatgcagaccgccaagacccaaccccgggaagagcagttctcaggcacctacagggtggtgagtgtgttgcccatcggccaccaggactggctgaaggggaagcaattcacatgcaaggttaataacaaggccctgcccagccccatcgagaggaccatcagcaaggccaggggccaggcccaccagccatctgtgtacgtgctgcccccatctagggaggaactgagcaagaacacagtcagccttacttgcctgatcaaggacttcttcccaccggacatagacgtggagtggcagagtaacggccagcaggagcccgagagcaagtataggaccacaccgccccaactggacgaggacggaagctacttcctctacagcaaattgagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgcgccgtgatgcacgaggctttgcataaccactacacccaggagagcctgtc ccacagccccggatag,In embodiments, the cDNA comprises the nucleic acid sequence(SEQ ID NO: 37) atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggaactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaaatgtcctccgcctgagatgctgggtggccctagcatcttcatcttcccgcccaagcccaaggatactctgtccattagcaggacccccgaggtgacctgcctggtggtggacctggggccagacgactctgacgtgcagatcacctggttcgtagacaacacccaggtttacactgccaagaccagtcccagggaggagcagttcaacagcacatacagggtggtgagcgttctgcccatcctgcaccaggactggctgaaaggcaaagagttcaagtgtaaggtgaacagcaagagcctgcccagccccattgaaaggaccatcagcaaggacaagggccagccgcacgagccccaagtctacgtgctgcccccagcacaggaagagctgagcaggaacaaggttagcgtgacatgcctgatcgagggtttctaccccagcgacatcgccgtggagtgggaaatcaccggccaacccgagcccgagaacaactacaggaccactccgccgcaactggacagcgacgggacctacttcttgtatagcaggctgagcgtggaccggagcaggtggcagaggggcaacacctacacttgcagcgtgagccacgaggccttgcacagccaccacactcagaagagtctgac ccagagcccgggatag.In embodiments, the cDNA comprises the nucleic acid sequence(SEQ ID NO: 39) atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggcactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaaatgtcctccgcctgagatgctgggtggccctagcatcttcatcttcccgcccaagcccaaggatactctgtccattagcaggacccccgaggtgacctgcctggtggtggacctggggccagacgactctgacgtgcagatcacctggttcgtagacaacacccaggtttacactgccaagaccagtcccagggaggagcagttcagcagcacatacagggtggtgagcgttctgcccatcctgcaccaggactggctgaaaggcaaagagttcaagtgtaaggtgaacagcaagagcctgcccagccccattgaaaggaccatcagcaaggacaagggccagccgcacgagccccaagtctacgtgctgcccccagcacaggaagagctgagcaggaacaaggttagcgtgacatgcctgatcgagggtttctaccccagcgacatcgccgtggagtgggaaatcaccggccaacccgagcccgagaacaactacaggaccactccgccgcaactggacagcgacgggacctacttcttgtatagcaggctgagcgtggaccggagcaggtggcagaggggcaacacctacacttgcagcgtgagccacgaggccttgcacagccaccacactcagaagagtctgac ccagagcccgggatag.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic representation of an exemplary insulin-Fcfusion protein homodimer.

FIG. 2 shows average % fasting blood glucose levels from Day 0 to Day 3for N=3 dogs dosed intravenously on Day 0 at 0.2 mg/kg with thehomodimer of SEQ ID NO: 42.

FIG. 3 illustrates a side-by-side sequence comparison of SEQ ID NOs: 42,44, 46, 48, and 50. “*” represents complete homology across allsequences at a given sequence position, while “:”, “.” or spaces referto conservative, moderate, or very different amino acid mutations acrossthe sequences at a given sequence position respectively.

FIG. 4 illustrates a side-by-side sequence comparison of SEQ ID NOs: 42,52, 54, and 56. “*” represents complete homology across all sequences ata given sequence position, while “:”, “.” or spaces refer toconservative, moderate, or very different amino acid mutations acrossthe sequences at a given sequence position respectively.

FIG. 5 shows average % fasting blood glucose levels from Day 0 to Day 7for N=3 dogs dosed intravenously on Day 0 at 0.2 mg/kg with thehomodimer of SEQ ID NO: 52.

FIG. 6 shows average % fasting blood glucose levels from Day 0 to Day 7for N=6 dogs dosed subcutaneously on Day 0 at 0.33 mg/kg with thehomodimer of SEQ ID NO: 52.

FIG. 7 shows the average anti-drug antibody titer (μg/mL) for N=3 dogsdosed subcutaneously on Day 0 (0.30 mg/kg), Day 28 (0.33 mg/kg), Day 35(0.33 mg/kg), Day 42 (0.50 mg/kg), Day 49 (1.00 mg/kg) and Day 56 (1.00mg/kg) with the homodimer of SEQ ID NO: 52.

FIG. 8 illustrates a side-by-side sequence comparison of SEQ ID NOs: 58,60, 62, and 64. “*” represents complete homology across all sequences ata given sequence position, while “:”, “.” or spaces refer toconservative, moderate, or very different amino acid mutations acrossthe sequences at a given sequence position respectively.

FIG. 9 shows the average anti-drug antibody titer (μg/mL) for N=1 dogdosed subcutaneously on Day 0 (0.33 mg/kg), Day 7 (0.50 mg/kg), Day 14(0.50 mg/kg), and Day 21 (0.50 mg/kg) with the homodimer of SEQ ID NO:64.

FIG. 10 shows the average anti-drug antibody titer (μg/mL) for N=1 dogsdosed subcutaneously on Day 0 (0.33 mg/kg) and Day 14 (0.16 mg/kg) withthe homodimer of SEQ ID NO: 66.

FIG. 11 shows average % fasting blood glucose levels from Day 0 to Day 7for N=2 dogs dosed subcutaneously on Day 0 at 0.33 mg/kg with thehomodimer of SEQ ID NO: 66.

FIG. 12 illustrates a side-by-side sequence comparison of SEQ ID NOs:66, 68, 70, 72, 74 and 76. “*” represents complete homology across allsequences at a given sequence position, while “:”, “.” or spaces referto conservative, moderate, or very different amino acid mutations acrossthe sequences at a given sequence position respectively.

FIG. 13 illustrates a side-by-side sequence comparison of SEQ ID NOs:66, 78, 80, 82, and 84. “*” represents complete homology across allsequences at a given sequence position, while “:”, “.” or spaces referto conservative, moderate, or very different amino acid mutations acrossthe sequences at a given sequence position respectively.

FIG. 14 illustrates a side-by-side sequence comparison of SEQ ID NOs:66, 76 and 86. “*” represents complete homology across all sequences ata given sequence position, while “:”, “.” or spaces refer toconservative, moderate, or very different amino acid mutations acrossthe sequences at a given sequence position respectively.

FIG. 15 illustrates a side-by-side sequence comparison of SEQ ID NOs:66, 82, 84 and 88. “*” represents complete homology across all sequencesat a given sequence position, while “:”, “.” or spaces refer toconservative, moderate, or very different amino acid mutations acrossthe sequences at a given sequence position respectively.

FIG. 16 illustrates a side-by-side sequence comparison of SEQ ID NOs:32, 34, 66, 90, 92 and 94. “*” represents complete homology across allsequences at a given sequence position, while “:”, “.” or spaces referto conservative, moderate, or very different amino acid mutations acrossthe sequences at a given sequence position respectively.

FIG. 17 shows % fasting blood glucose levels from Day 0 to Day 7 for N=1dog dosed subcutaneously on Day 0 at 0.16 mg/kg with the homodimer ofSEQ ID NO: 34.

FIG. 18 shows the anti-drug antibody titer (μg/mL) for N=1 dog dosedsubcutaneously on Day 0 (0.16 mg/kg), Day 14 (0.16 mg/kg), Day 28 (0.16mg/kg), and Day 42 (0.16 mg/kg) with the homodimer of SEQ ID NO: 34.

FIG. 19 shows % fasting blood glucose levels from Day 0 to Day 7 for N=1dog dosed subcutaneously on Day 0 at 0.33 mg/kg with the homodimer ofSEQ ID NO: 32.

FIG. 20 shows % fasting blood glucose levels from Day 0 to Day 60 forN=1 dog dosed subcutaneously on Day 0 (0.33 mg/kg), Day 15 (0.16 mg/kg),Day 31 (0.16 mg/kg) and Day 45 (0.15 mg/kg) with the homodimer of SEQ IDNO: 32.

FIG. 21 shows the anti-drug antibody titer (μg/mL) for N=1 dogs dosedsubcutaneously on Day 0 (0.33 mg/kg), Day 15 (0.16 mg/kg), Day 31 (0.16mg/kg) and Day 45 (0.15 mg/kg) with the homodimer of SEQ ID NO: 32.

FIG. 22 shows % fasting blood glucose levels from Day 0 to Day 7 for N=1dog dosed subcutaneously on Day 0 at 0.16 mg/kg with the homodimer ofSEQ ID NO: 96.

FIG. 23 shows % fasting blood glucose levels from Day 0 to Day 7 for N=1dog dosed subcutaneously on Day 0 at 0.16 mg/kg with the homodimer ofSEQ ID NO: 98.

FIG. 24 illustrates a side-by-side sequence comparison of SEQ ID NOs:102 and 104. “*” represents complete homology across all sequences at agiven sequence position, while “:”, “.” or spaces refer to conservative,moderate, or very different amino acid mutations across the sequences ata given sequence position respectively.

FIG. 25 shows % fasting blood glucose levels from Day 0 to Day 7 for N=1dog dosed subcutaneously on Day 0 at 0.16 mg/kg with the homodimer ofSEQ ID NO: 102, and % fasting blood glucose levels from Day 0 to Day 7for N=1 dog dosed subcutaneously on Day 0 at 0.16 mg/kg with thehomodimer of SEQ ID NO: 104.

FIG. 26 shows % fasting blood glucose levels from Day 0 to Day 7 for N=1dog dosed subcutaneously c with the homodimer of SEQ ID NO: 36 inaddition to the times that the dog was given food.

FIG. 27 shows average % fasting blood glucose levels from Day 0 to Day 7for N=3 cats dosed subcutaneously on Day 0 at 0.8 mg/kg with thehomodimer of SEQ ID NO: 106.

FIG. 28 illustrates a side-by-side sequence comparison of SEQ ID NOs:106, 108, 110 and 112. “*” represents complete homology across allsequences at a given sequence position, while “:”, “.” or spaces referto conservative, moderate, or very different amino acid mutations acrossthe sequences at a given sequence position respectively.

FIG. 29 shows the average anti-drug antibody titer (μg/mL) for N=3 catsdosed subcutaneously on Day 0 (0.8 mg/kg), Day 28 (0.6 mg/kg), Day 35(0.6 mg/kg), Day 42 (0.6 mg/kg) and Day 48 (0.8 mg/kg) with thehomodimer of SEQ ID NO: 106.

FIG. 30 illustrates a side-by-side sequence comparison of SEQ ID NOs:108, 114, 116 and 118. “*” represents complete homology across allsequences at a given sequence position, while “:”, “.” or spaces referto conservative, moderate, or very different amino acid mutations acrossthe sequences at a given sequence position respectively.

FIG. 31 illustrates a side-by-side sequence comparison of SEQ ID NOs:106, 112, and 122. “*” represents complete homology across all sequencesat a given sequence position, while “:”, “.” or spaces refer toconservative, moderate, or very different amino acid mutations acrossthe sequences at a given sequence position respectively.

FIG. 32 shows % fasting blood glucose levels from Day 0 to Day 7 for N=1cat dosed subcutaneously on Day 0 (0.16 mg/kg) with the homodimer of SEQID NO: 122.

FIG. 33 shows % fasting blood glucose levels from Day 0 to Day 7 for N=1cat dosed subcutaneously on Day 0 (0.16 mg/kg) with the homodimer of SEQID NO: 38, in addition to the times that the cat was given food.

FIG. 34 shows the anti-drug antibody titer (μg/mL) for N=1 cat dosedsubcutaneously on Day 0 (0.16 mg/kg), Day 14 (0.16 mg/kg), Day 28 (0.11mg/kg), and Day 42 (0.09 mg/kg) with the homodimer of SEQ ID NO: 38.

FIG. 35 shows % fasting blood glucose levels from Day 0 to Day 7 for N=1cat dosed subcutaneously on Day 0 (0.16 mg/kg) with the homodimer of SEQID NO: 124.

FIG. 36 shows average % fasting blood glucose levels from Day 0 to Day 7for N=3 cats dosed subcutaneously on Day 0 (0.10 mg/kg) with thehomodimer of SEQ ID NO: 40.

FIG. 37 shows average % fasting blood glucose levels from Day 7 to Day14 for N=3 cats dosed subcutaneously on Day 7 (0.20 mg/kg) with thehomodimer of SEQ ID NO: 40.

FIG. 38 illustrates the “full aa sequence” of a fusion protein (SEQ IDNO: 32) and its corresponding nucleic acid sequence (SEQ ID NO: 31).

FIG. 39 illustrates the “full aa sequence” of a fusion protein (SEQ IDNO: 34) and its corresponding nucleic acid sequence (SEQ ID NO: 33).

FIG. 40 illustrates the “full aa sequence” of a fusion protein (SEQ IDNO: 36) and its corresponding nucleic acid sequence (SEQ ID NO: 35).

FIG. 41 illustrates the “full aa sequence” of a fusion protein (SEQ IDNO: 38) and its corresponding nucleic acid sequence (SEQ ID NO: 37).

FIG. 42 illustrates the “full aa sequence” of a fusion protein (SEQ IDNO: 40) and its corresponding nucleic acid sequence (SEQ ID NO: 39).

DETAILED DESCRIPTION

An insulin treatment that requires less frequent dosing (e.g.,once-weekly injections) would be less burdensome on the owners, leadingto better compliance, fewer instances of euthanasia, and better outcomesfor the pets. For a given species (e.g., dog or cat), a moleculesuitable for an ultra-long acting treatment for diabetes should bemanufacturable in mammalian cells, for example human embryonic kidney(HEK, e.g. HEK293) cells, with an acceptable titer of the desiredhomodimer product (e.g., greater than 50 mg/L homodimer titer fromtransiently transfected HEK cells, greater than 75 mg/L from transientlytransfected from HEK cells, greater than 100 mg/L from transientlytransfected HEK cells, etc.). Only candidates with a homodimer titer ofgreater than 50 mg/L are considered useful in the present invention,because experience has demonstrated that homodimer titers less than thislevel will not likely result in commercial production homodimer titersin Chinese hamster ovary (CHO) cells that meet the stringently lowmanufacturing cost requirements for veterinary products. In addition,the molecule must bind the insulin receptor with an appreciable affinity(e.g., IC50 less than 5000 nM, IC50 less than 4000 nM, IC50 less than3000 nM, IC50 less than 2500 nM, etc.) as measured in the 4° C. IM-9insulin receptor binding assay. Based on experience, only moleculesexhibiting insulin receptor activity IC50 values less than 5000 nM aredeemed likely to exhibit the requisite bioactivity in the targetspecies. The molecule must also demonstrate sustained bioactivity invivo (e.g., demonstrate glucose lowering activity greater than about 2hours, 6 hours, 9 hours, 12 hours, 18 hours, 1 day, 1.5 days, 2 days,2.5 days, 3 days, 4 days, 5 days, 6 days, 7 days, or longer) to justifyless frequent dosing. The molecule must also demonstrate prolongedsystem residence time in the target animal (e.g., the serum half-lifemust be greater than 3 days, or longer). The bioactive potency andduration of the bioactivity may be quantitatively represented bycalculating the area over the percent fasting blood glucose (% FBGL)curve normalized to a given dose in mg/kg (NAOC) with units of %FBGL·days·kg/mg as described in Example 11. The NAOC increases with agreater drop in % FBGL, which is the case where the moleculedemonstrates increased bioactivity, and when the % FBGL takes longer toreturn to 100%, which is the case where the insulin-Fc fusion proteindemonstrates increased duration of action. To be useful as describedherein, a molecule must demonstrate a sufficiently high NAOC value (e.g.preferably NAOC greater than 150% FBGL·days·kg/mg, more preferably NAOCgreater than 200% FBGL·days·kg/mg, and even more preferably NAOC greaterthan 250% FBGL·days·kg/mg). Based on experience, at NAOC values greaterthan 150% FBGL·days·kg/mg, the dose requirements in the target specieswill be sufficiently low so as to reach an acceptable treatment cost.Lastly, to be useful for treating a chronic disease such as diabetes,the molecule must not induce the production of anti-drug antibodies,especially antibodies that neutralize the bioactivity of the moleculeafter repeated dosing. Therefore, the molecule must demonstrate similarduration and extent of bioactivity (i.e., NAOC) after multiple repeateddoses in the target animal (e.g., the ratio of the NAOC after the thirdweekly subcutaneous injection to the NAOC after the first weeklysubcutaneous injection of the molecule (i.e., the NAOC ratio (NAOCR)after the third dose) is in order of preference greater than 0.50,greater than 0.60, greater than 0.70, greater than 0.80, or greater than0.90 or more).

Proposed ultra-long acting insulin treatments for human clinical usecomprise an insulin-Fc fusion protein making use of a human Fc fragmentto prolong their action in vivo. As a human Fc fragment is expected tobe immunogenic and therefore capable of inducing the production ofanti-drug antibodies in companion animals (e.g., dogs or cats), thehuman Fc fragment must be replaced with a species-specific (e.g., canineor feline) Fc fragment. However, it was found rather unexpectedly that asimple exchange between the human Fc fragment and the species-specific(e.g., canine or feline) Fc fragment did not yield a product with anacceptable homodimer titer (e.g., a homodimer titer greater than 50mg/L) or a sufficiently high NAOC value (e.g., a NAOC greater than 150%FBGL·days·kg/mg). For example, in some cases only a specific isotype(e.g., canine IgGB or feline IgG1b) for the Fc fragment resulted in aninsulin-Fc fusion protein with a high enough homodimer titer (e.g., ahomodimer titer greater than 50 mg/L) and an acceptably high NAOC value(e.g., a NAOC greater than 150% FBGL·days·kg/mg). In other cases,specific amino acids of the insulin polypeptide were found to beimmunogenic in the target species thereby requiring site-directedmutations to find the relatively small number of embodiments that wereboth non-immunogenic and bioactive in the target species with acceptablyhigh NAOC values (e.g., NAOC values greater than 150% FBGL·days·kg/mg)and NAOCR values after the third weekly subcutaneous dose that weregreater than 0.5. In further cases, when the Fc fragments were mutatedto prevent glycosylation and thereby further reduce the immunogenicityof the insulin-Fc fusion proteins, it was discovered unexpectedly thatonly specific amino acid mutations in the Fc fragment led to the desiredhomodimer titers (e.g., homodimer titers greater than 50 mg/L) and NAOCvalues (e.g., NAOC greater values than 150% FBGL·days·kg/mg).Furthermore, it was discovered that an additional mutation in theinsulin component was required to produce these Fc-mutated,non-glycosylated insulin Fc-fusion proteins with the desired homodimertiters (e.g., homodimer titers greater than 50 mg/L) and NAOC values(e.g., NAOC greater values than 150% FBGL·days·kg/mg), while alsoachieving NAOCR values after the third weekly subcutaneous dose thatwere greater than 0.5. Provided herein, therefore, are manufacturable,high purity, long-acting, bioactive, non-immunogenic insulin-Fc fusionproteins with acceptably high homodimer titers (e.g., homodimer titersgreater than 50 mg/L), NAOC values (e.g., NAOC values greater than 150%FBGL·days·kg/mg), and NAOCR values after the third weekly subcutaneousdose greater than 0.5, suitable for the treatment of diabetes incompanion animals (e.g., dogs or cats), each of which comprises aninsulin polypeptide, an Fc fragment, and a linker between the insulinpolypeptide and the Fc fragment.

Definitions

As used herein, the articles “a” and “an” refer to one or more than one,e.g., to at least one, of the grammatical object of the article. The useof the words “a” or “an” when used in conjunction with the term“comprising” herein may mean “one,” but it is also consistent with themeaning of “one or more,” “at least one,” and “one or more than one.”

As used herein, “about” and “approximately” generally mean an acceptabledegree of error for the quantity measured given the nature or precisionof the measurements. Exemplary degrees of error are within 20 percent(%), typically, within 10%, and more typically, within 5% of a givenrange of values.

As used herein, an amount of a molecule, compound, conjugate, orsubstance effective to treat a disorder (e.g., a disorder describedherein), “therapeutically effective amount,” or “effective amount”refers to an amount of the molecule, compound, conjugate, or substancewhich is effective, upon single or multiple dose administration(s) to asubject, in treating a subject, or in curing, alleviating, relieving orimproving a subject with a disorder (e.g., a disorder described herein)beyond that expected in the absence of such treatment.

As used herein, the term “analog” refers to a compound or conjugate(e.g., a compound or conjugate as described herein, e.g., insulin)having a chemical structure similar to that of another compound orconjugate but differing from it in at least one aspect.

As used herein, the term “antibody” or “antibody molecule” refers to animmunoglobulin molecule (Ig), immunologically active portions of animmunoglobulin (Ig) molecule, i.e., a molecule that contains an antigenbinding site that specifically binds, e.g., immunoreacts with, anantigen. As used herein, the term “antibody domain” refers to a variableor constant region of an immunoglobulin. As used herein, the term“antibody domain” refers to a variable or constant region of animmunoglobulin. It is documented in the art that antibodies compriseseveral classes, for example IgA, IgM, or IgG in the case of mammals(e.g., humans and felines). Classes of immunoglobulins can be furtherclassified into different isotypes, such as IgGA, IgGB, IgGC, and IgGDfor canines, or IgG1a, IgG1b, and IgG2 for felines. Those skilled in theart will recognize that immunoglobulin isotypes of a givenimmunoglobulin class will comprise different amino acid sequences,structures, and functional properties from one another (e.g., differentbinding affinities to Fc(gamma) receptors). “Specifically binds” or“immunoreacts with” means that the antibody reacts with one or moreantigenic determinants of the desired antigen and has a lower affinityfor other polypeptides, e.g., does not react with other polypeptides.

As used herein, the term “area-under-the-curve” or “AUC” refers to theintegrated area under the % FBGL vs. time curve for a subject after agiven dose of an insulin-Fc fusion protein is administered. As usedherein, the term “area-over-the curve” or “AOC” is used as a measure ofthe biological potency of an insulin-Fc fusion protein such that the AOCequals the difference between the total possible area under the % FBGLvs. time curve and the AUC value. As used herein, the “normalizedarea-over-the curve,” “normalized AOC,” or “NAOC” is the AOC valuedivided by the actual dose of insulin-Fc fusion protein administered. Asused herein, the term “normalized AOC ratio” or “NAOCR” is the ratio ofthe NAOC resulting from a particular administration of an insulin-Fcfusion protein to the NAOC resulting from the first administration of aninsulin-Fc fusion protein in a series of administrations. The NAOCR thusprovides a measure of the change in biological activity of an insulin-Fcfusion protein after repeated administrations.

As used herein, the term “bioactivity,” “activity,” “biologicalactivity,” “potency,” “bioactive potency,” or “biological potency”refers to the extent to which an insulin-Fc fusion protein activates theinsulin receptor and/or exerts a reduction in blood glucose levels in atarget subject. As used herein, “in vitro activity” or “insulin receptoractivity” refers to the affinity with which an insulin-Fc fusion proteinbinds to the insulin receptor and is typically measured by theconcentration at which an insulin-Fc fusion protein displaces half of aninsulin reference standard from the insulin receptor in a competitivebinding assay (i.e., IC50). As used herein, “in vivo activity” refers tothe extent and duration of reduction in a target subject's fasting bloodglucose level after administration of an insulin-Fc fusion protein.

As used herein, the term “biosynthesis,” “recombinant synthesis,” or“recombinantly made” refers to the process by which an insulin-Fc fusionprotein is expressed within a host cell by transfecting the cell with anucleic acid molecule (e.g., vector) encoding the insulin-Fc fusionprotein (e.g., where the entire insulin-Fc fusion protein is encoded bya single nucleic acid molecule). Exemplary host cells include mammaliancells, e.g., HEK293 cells or CHO cells. The cells can be cultured usingstandard methods in the art and the expressed insulin-Fc fusion proteinmay be harvested and purified from the cell culture using standardmethods in the art.

As used herein, the term “cell surface receptor” refers to a moleculesuch as a protein, generally found on the external surface of themembrane of a cell and which interacts with soluble molecules, e.g.,molecules that circulate in the blood supply. In some embodiments, acell surface receptor may include a hormone receptor (e.g., an insulinhormone receptor or insulin receptor (IR)) or an Fc receptor which bindsto an Fc fragment or the Fc region of an antibody (e.g. an Fc(gamma)receptor, for example Fc(gamma) receptor I, or an Fc neonatal receptor,for example FcRn). As used herein, “in vitro activity” or “Fc(gamma)receptor activity” or “Fc(gamma) receptor binding” or “FcRn receptoractivity” or “FcRn binding” refers to the affinity with which aninsulin-Fc fusion protein binds to the Fc receptor (e.g. Fc(gamma)receptor or FcRn receptor) and is typically measured by theconcentration of an insulin-Fc fusion protein that causes the insulin-Fcfusion protein to reach half of its maximum binding (i.e., EC50 value)as measured on an assay (e.g., an enzyme-linked immunosorbent assay(ELISA) assay) using OD 450 nm values as measured on a microplatereader.

As used herein, the term “fasting blood glucose level” or “FBGL” refersto the average blood glucose level in a target subject at the end of aperiod during which no food is administered and just prior to the timeat which an insulin-Fc fusion protein is administered. As used herein,the term “percent fasting blood glucose level,” “% fasting blood glucoselevel,” or “% FBGL” refers to the ratio of a given blood glucose levelto the fasting blood glucose level multiplied by 100.

As used herein, the term “immunogenic” or “immunogenicity” refers to thecapacity for a given molecule (e.g., an insulin-Fc fusion protein of thepresent invention) to provoke the immune system of a target subject suchthat after repeated administrations of the molecule, the subjectdevelops antibodies capable of specifically binding the molecule (i.e.,anti-drug antibodies). As used herein, the terms “neutralizing,”“neutralizing antibodies”, or “neutralizing anti-drug antibodies” referto the capacity for antibodies to interfere with the compound'sbiological activity in the target subject. As used herein, the term“immunogenic epitopes,” ‘immunogenic hot spots,” or “hot spots” refersto the mutations or epitopes of a given molecule (e.g., an insulin-Fcfusion protein of the present invention) that are responsible formoderate or strong binding of the anti-drug antibodies.

As used herein, the term “insulin reference standard” is any one of: (i)a naturally occurring insulin from a mammal (e.g., a human, a dog, or acat); (ii) an insulin polypeptide that does not comprise an Fc fragment;or (iii) a standard of care insulin (e.g., a commercially availableinsulin).

As used herein, the term “monomer” refers to a protein or a fusionprotein comprising a single polypeptide. In embodiments, the “monomer”is a protein or a fusion protein, e.g., a single polypeptide, comprisingan insulin polypeptide and an Fc fragment polypeptide, wherein theinsulin and Fc fragment polypeptides are joined by peptide bonds to formthe single polypeptide. In embodiments, the monomer is encoded by asingle nucleic acid molecule.

As used herein, “N-terminus” refers to the start of a protein orpolypeptide that is initiated by an amino acid containing a free aminegroup that is the alpha-amino group of the amino acid (e.g. the freeamino that is covalently linked to one carbon atom that is locatedadjacent to a second carbon atom, wherein the second carbon atom is partof the carbonyl group of the amino acid). As used herein, “C-terminus”refers to the end of a protein or polypeptide that is terminated by anamino acid containing a carboxylic acid group, wherein the carbon atomof the carboxylic acid group is located adjacent to the alpha-aminogroup of the amino acid.

As used herein, “pharmacodynamics” or “PD” generally refers to thebiological effects of an insulin-Fc fusion protein in a subject.Specifically, herein the PD refers to the measure of the reduction infasting blood glucose level over time in a subject after theadministration of an insulin-Fc fusion protein.

As used herein, “pharmacokinetics” or “PK” generally refers to thecharacteristic interactions of an insulin-Fc fusion protein and the bodyof the subject in terms of its absorption, distribution, metabolism, andexcretion. Specifically, herein the PK refers to the concentration of aninsulin-Fc fusion protein in the blood or serum of a subject at a giventime after the administration of the insulin-Fc fusion protein. As usedherein, “half-life” refers to the time taken for the concentration ofinsulin-Fc fusion protein in the blood or serum of a subject to reachhalf of its original value as calculated from a first order exponentialdecay model for drug elimination. Insulin-Fc fusion proteins withgreater “half-life” values demonstrate greater duration of action in thetarget subject.

The terms “sequence identity” “sequence homology” “homology” or“identical” in amino acid or nucleotide sequences as used hereindescribes that the same nucleotides or amino acid residues are foundwithin the variant and reference sequences when a specified, contiguoussegment of the nucleotide sequence or amino acid sequence of the variantis aligned and compared to the nucleotide sequence or amino acidsequence of the reference sequence. Methods for sequence alignment andfor determining identity between sequences are known in the art,including the use of Clustal Omega, which organizes, aligns, andcompares sequences for similarity, wherein the software highlights eachsequence position and compares across all sequences at that position andassigns one of the following scores: an “*” (asterisk) for sequencepositions which have a single, fully conserved residue, a “:” (colon)indicates conservation between groups of strongly similar propertieswith scoring greater than 0.5 in the Gonnet PAM 250 matrix, and a “.”(period) indicates conservation between groups of weakly similarproperties with scoring less than or equal to 0.5 in the Gonnet PAM 250matrix, a “-” (dash) indicates a sequence gap, meaning that no localhomology exists within a particular set of comparisons within a certainrange of the sequences, and an empty space “ ” indicates little or nosequence homology for that particular position across the comparedsequences. See, for example Ausubel et al., eds. (1995) CurrentProtocols in Molecular Biology, Chapter 19 (Greene Publishing andWiley-Interscience, New York); and the ALIGN program (Dayhoff (1978) inAtlas of Polypeptide Sequence and Structure 5: Suppl. 3 (NationalBiomedical Research Foundation, Washington, D.C.). With respect tooptimal alignment of two nucleotide sequences, the contiguous segment ofthe variant nucleotide sequence may have additional nucleotides ordeleted nucleotides with respect to the reference nucleotide sequence.Likewise, for purposes of optimal alignment of two amino acid sequences,the contiguous segment of the variant amino acid sequence may haveadditional amino acid residues or deleted amino acid residues withrespect to the reference amino acid sequence. In some embodiments, thecontiguous segment used for comparison to the reference nucleotidesequence or reference amino acid sequence will comprise at least 6, 10,15, or 20 contiguous nucleotides, or amino acid residues, and may be 30,40, 50, 100, or more nucleotides or amino acid residues. Corrections forincreased sequence identity associated with inclusion of gaps in thevariant's nucleotide sequence or amino acid sequence can be made byassigning gap penalties. Methods of sequence alignment are known in theart.

In embodiments, the determination of percent identity or “homology”between two sequences is accomplished using a mathematical algorithm.For example, the percent identity of an amino acid sequence isdetermined using the Smith-Waterman homology search algorithm using anaffine 6 gap search with a gap open penalty of 12 and a gap extensionpenalty of 2, BLOSUM matrix 62. The Smith-Waterman homology searchalgorithm is described in Smith and Waterman (1981) Adv. Appl. Math2:482-489, herein incorporated by reference. In embodiments, the percentidentity of a nucleotide sequence is determined using the Smith-Watermanhomology search algorithm using a gap open penalty of 25 and a gapextension penalty of 5. Such a determination of sequence identity can beperformed using, for example, the DeCypher Hardware Accelerator fromTimeLogic.

As used herein, the term “homology” is used to compare two or moreproteins by locating common structural characteristics and commonspatial distribution of, for instance, beta strands, helices, and folds.Accordingly, homologous protein structures are defined by spatialanalyses. Measuring structural homology involves computing thegeometric-topological features of a space. One approach used to generateand analyze three-dimensional (3D) protein structures is homologymodeling (also called comparative modeling or knowledge-based modeling)which works by finding similar sequences on the basis of the fact that3D similarity reflects 2D similarity. Homologous structures do not implysequence similarity as a necessary condition.

As used herein, the terms “subject” and “patient” are intended toinclude canine and feline animals. Exemplary canine and feline subjectsinclude dogs and cats having a disease or a disorder, e.g., diabetes oranother disease or disorder described herein, or normal subjects.

As used herein, the term “titer” or “yield” refers to the amount of afusion protein product (e.g., an insulin-Fc fusion protein describedherein) resulting from the biosynthesis (e.g., in a mammalian cell,e.g., in a HEK293 cell or CHO cell) per volume of the cell culture. Theamount of product may be determined at any step of the productionprocess (e.g., before or after purification), but the yield or titer isalways stated per volume of the original cell culture. As used herein,the term “product yield” or “total protein yield” refers to the totalamount of insulin-Fc fusion protein expressed by cells and purified viaat least one affinity chromatography step (e.g. Protein A or Protein G)and includes monomers of insulin-Fc fusion protein, homodimers ofinsulin-Fc fusion protein, and higher-order molecular aggregates ofhomodimers of insulin-Fc fusion protein. As used herein, the term“percent homodimer” or “% homodimer” refers to the proportion of afusion protein product (e.g., an insulin-Fc fusion protein describedherein) that is the desired homodimer. As used herein, the term“homodimer titer” refers to the product of the % homodimer and the totalprotein yield after Protein A purification step reported per volume ofthe cell culture.

As used herein, the terms “treat” or “treating” a subject having adisease or a disorder refer to subjecting the subject to a regimen, forexample the administration of a fusion protein such as a fusion proteindescribed herein, such that at least one symptom of the disease ordisorder is cured, healed, alleviated, relieved, altered, remedied,ameliorated, or improved. Treating includes administering an amounteffective to alleviate, relieve, alter, remedy, ameliorate, improve oraffect the disease or disorder, or the symptoms of the disease ordisorder. The treatment may inhibit deterioration or worsening of asymptom of a disease or disorder.

Insulin-Fc Fusion Protein Components and Structure

The present disclosure relates to a composition of a fusion protein(i.e., an insulin-Fc fusion protein) comprising an insulin polypeptidelinked via a peptide linker to a species-specific Fc fragment, and itsuse to treat diabetes in companion animals (e.g., dogs or cats). As usedherein, the terms “fusion protein” and “insulin-Fc fusion protein” referto a protein comprising more than one part, for example from differentsources (different proteins, polypeptides, cells, etc.), that arecovalently linked through peptide bonds. The insulin-Fc fusion proteinsare covalently linked by (i) connecting the genes that encode for eachpart into a single nucleic acid molecule and (ii) expressing in a hostcell (e.g., HEK or CHO) the protein for which the nucleic acid moleculeencodes as follows: (N-terminus)--insulin polypeptide--linker--Fcfragment--(C-terminus). The fully recombinant synthesis approach ispreferred over methods in which the insulin polypeptide and Fc fragmentsare synthesized separately and then chemically conjugated. The chemicalconjugation step and subsequent purification process increase themanufacturing complexity, reduce product yield, and increase cost.

As used herein, the term “dimer” refers to a protein or a fusion proteincomprising two polypeptides linked covalently. In embodiments, twoidentical polypeptides are linked covalently (e.g., via disulfide bonds)forming a “homodimer” (diagrammatically represented in FIG. 1).Disulfide bonds are shown as dotted lines in FIG. 1; total number ofdisulfide bonds in actuality may be greater or less than the numbershown in FIG. 1. In embodiments, the homodimer is encoded by a singlenucleic acid molecule, wherein the homodimer is made recombinantlyinside a cell by first forming insulin-Fc fusion protein monomers and bythen assembling two identical insulin-Fc fusion protein monomers intothe homodimer upon further processing inside the cell.

As used herein, the terms “multimer,” “multimeric,” or “multimericstate” refer to non-covalent, associated forms of Fc fusion proteindimers that may be in equilibrium with Fc fusion protein dimers or mayact as permanently aggregated versions of Fc fusion protein dimers(e.g., dimers of Fc fusion protein homodimers, trimers of Fc fusionprotein homodimers, tetramers of Fc fusion protein homodimers, or higherorder aggregates containing five or more Fc fusion protein homodimers).It may be expected that multimeric forms of Fc fusion proteins may havedifferent physical, stability, or pharmacologic activities from that ofthe insulin-Fc fusion protein homodimers.

Insulin Polypeptide

An insulin polypeptide may be, for example, an insulin or insulin analogproduced by β-cells in the islets of Langerhans within the pancreas.Insulin functions by regulating the absorption of glucose from theblood. Upon a stimulus, such as increased protein and glucose levels,insulin is released from β-cells and binds to the insulin receptor (IR),initiating a signal cascade that affects many aspects of mammalian(e.g., human, canine, or feline) metabolism. Disruption of this processis directly related to several diseases, notably diabetes, insulinoma,insulin resistance, metabolic syndromes, and polycystic ovary syndrome.Insulin analogs of the present disclosure may be related to thestructure of insulin yet contain one or more modifications. In someembodiments, the insulin analog comprises at least one amino acidsubstitution, deletion, addition or chemical modification relative toinsulin, which may impact a particular feature or characteristic of theinsulin-Fc fusion protein. For example, the modifications or alterationsdescribed herein may impact the structure, stability, pH sensitivity,bioactivity, or binding affinity of the insulin-Fc fusion protein to acell surface receptor (e.g. an insulin hormone receptor) relative to areference standard.

The amino acid sequence of insulin is strongly conserved throughoutevolution, particularly in vertebrates. For example, native canineinsulin differs by only one amino acid from human insulin, and nativefeline insulin differs by just four amino acids from human insulin. Asused herein, the terms “B-chain”, “C-peptide” or “C-chain”, and“A-chain” refer to the peptide segments of an insulin polypeptide asillustrated in FIG. 1. Insulin is a 51 amino acid hormone containing twopeptide chains (i.e., a B-chain and an A-chain) connected via disulfidebonds (e.g., disulfide bonds formed by one or more B-chain cysteine sidechain thiols and one or more A-chain cysteine side chain thiols). TheA-chain of insulin is 21 amino acids in length and the B-chain ofinsulin is 30 amino acids in length. In the native form of insulin, theA-chain contains one intrachain disulfide bond formed by two A-chaincysteine side chain thiols. For reference purposes, the sequences forthe human insulin A-chain of SEQ ID NO: 1 and the human insulin B-chainof and SEQ ID NO: 2 are shown below:

(SEQ ID NO: 1) FVNQHLCGSHLVEALYVCGERGFFYTPKT (SEQ ID NO: 2)GIVEQCCTSICSLYQLENYCN

As used herein, the term “insulin” or “insulin polypeptide” encompassesmature insulin, preproinsulin, proinsulin, and naturally occurringinsulin, or analogs thereof. In embodiments, an insulin polypeptide canbe a full-length insulin polypeptide or a fragment thereof. Inembodiments, an insulin polypeptide can comprise one or more fragmentsfrom mature insulin, preproinsulin, proinsulin, or naturally occurringinsulin.

Insulin is normally constructed as aN-terminus--B-chain:C-chain:A-chain--C-terminus polypeptide, wherein theC-chain is cleaved in order to make it bioactive. For referencepurposes, the sequence of the entire human insulin molecule includingthe C-chain (i.e., human proinsulin) is shown below with the C-chainunderlined:

(SEQ ID NO: 3) FVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN

The transformation of the single-chain insulin polypeptide into abioactive two-chain polypeptide is normally accomplished within theβ-cells of the islets of Langerhans prior to glucose-stimulated insulinsecretion by two endoproteases, Type I endoproteases, PC1 and PC3, thatdisrupt the C peptide-B chain connection and PC2, and a Type IIendoprotease, that cleaves the C peptide-A chain bond at exactly theright sites. However, cell systems used for the biosynthesis oftherapeutic molecules such as insulin (e.g. bacteria, yeast, andmammalian (e.g. HEK and CHO) cell systems) do not possess this pathway,and therefore the transformation must take place after expression andharvesting of the single chain polypeptide using chemical or enzymaticmethods. All the known techniques for cleaving the C-chain afterexpression and harvesting rely on first modifying the C-chain such thatit terminates in a lysine just before the N-terminus of the A-chain.Then, using an enzyme selected from the trypsin or Lys-C families, whichclips peptide bonds specifically at the C-termini of lysine residues,the single chain-insulin polypeptide is cleaved at the C-terminal lysineof the C-chain and at the C-terminal lysine at the 29^(th) position fromthe N-terminus of the B-chain. In some cases, the resulting bioactivetwo-chain insulin is used without reattaching the clipped amino acid atthe 30^(th) position from the N-terminus of the B-chain, and in somecases the clipped amino acid at the 30^(th) position from the N-terminusof the B-chain is added back to the molecule using an additionalenzymatic method. Such a process works well with insulin, because itcontains only one lysine in its entire two chain polypeptide form.However, this process cannot be used on the insulin-Fc fusion proteinscontained herein, because all known Fc fragments contain multiple lysineresidues. The enzymatic cleavage process would, therefore, digest the Fcfragment into non-functional parts, thereby eliminating the ability ofthe Fc fragment to prolong the action of the insulin polypeptide invivo. Therefore, an insulin-Fc fusion protein of the present inventionmust comprise an insulin polypeptide that does not require C-chaincleavage and is therefore bioactive in its single chain form.

A number of bioactive single chain insulin polypeptides have beendescribed in the art. In all cases, the single chain insulinpolypeptides contain C-chains of specific length and composition as wellas A-chains and B-chains mutated at specific amino acid sites in orderto achieve electrostatic balance, prevent aggregation, and enhanceinsulin receptor (IR) binding and/or downstream signaling to achievebioactivity at levels comparable to that of the native two-chaininsulin. Herein, the location of mutations on peptide segments arenotated using the name of the segment (e.g., B-chain, C-chain, A-chain)and the number of the amino acid counting from the N-terminus of thesegment. For example, the notation “B16” refers to the 16^(th) aminoacid from the N-terminus of the amino acid sequence of the B-chain. Thenotation “A8” refers to the 8^(th) amino acid from the N-terminus of theA-chain. Furthermore, if an amino acid is mutated from its native formto a new amino acid at a particular location, the location is appendedwith the one letter amino acid code for the new amino acid. For example,B16A refers to an alanine mutation at the 16^(th) amino acid from theN-terminus of the amino acid sequence of the B-chain and A8H refers to ahistidine mutation at the 8^(th) amino acid from the N-terminus of theamino acid sequence of the A-chain.

In one example, a single chain insulin analog with a C-chain of thesequence GGGPRR and additional substitutions in the A-chain and B-chain(SEQ ID NO: 4) was developed by The Department of Biochemistry, CaseWestern Reserve University School of Medicine and the Department ofMedicine, University of Chicago (see Hua, Q.-x, Nakagawa, S. H., Jia,W., Huang, K., Phillips, N. B., Hu, S.-q., Weiss, M. A., (2008) J. Biol.Chem Vol. 283, No. 21 pp 14703-14716). In this example, at position 8 ofthe A-chain (i.e., A8), histidine is substituted for threonine; atposition 10 of the B-chain (i.e., B10), aspartic acid is substituted forhistidine; at position 28 of the B-chain (i.e., B28), aspartic acid issubstituted for proline; and at position 29 of the B-chain (i.e., B29),proline is substituted for lysine. SEQ ID NO: 4 is listed below witheach of the non-native amino acids underlined:

(SEQ ID NO: 4) FVNQHLCGSDLVEALYLVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCN

In embodiments, alanine may be substituted for tyrosine at position 16from the N-terminus of the B-chain (i.e., B16) in SEQ ID NO: 4 toproduce SEQ ID NO: 5, as an alanine substitution in this position isknown to be less capable of activating insulin-specific T cells (Alleva,D. G., Gaur, A., Jin, L., Wegmann, D., Gottlieb, P. A., Pahuja, A.,Johnson, E. B., Motheral, T., Putnam, A., Crowe, P. D., Ling, N.,Boehme, S. A., Conlon, P. J., (2002) Diabetes Vol. 51, No. 7 pp2126-2134). SEQ ID NO: 5 is listed below with each of the non-nativeamino acids underlined:

(SEQ ID NO: 5) FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCN

In some embodiments, it was unexpectedly discovered that specific aminoacids in SEQ ID NO: 4 and SEQ ID NO: 5 led to the development ofneutralizing anti-drug antibodies after repeated subcutaneous injectionsin the target animal (e.g., dog or cat). The anti-drug antibodies led toan unacceptable reduction in the NAOC after multiple injections (e.g., aNAOCR value after the third injection of less than 0.5), rendering theassociated insulin-Fc fusion proteins non-viable. Specifically, it wasdiscovered in the steps leading up to the invention of this disclosurethat the A8 mutation to histidine and the B10 mutation to aspartic acidaccounted for the vast majority of the anti-drug antibody specificityand thus represented immunogenic “hot spots” (e.g. immunogenic epitopes)on the insulin-polypeptide. Therefore, in preferred embodiments, theinsulin-polypeptide does not contain histidine at position A8 oraspartic acid at position B10 of the insulin polypeptide.

In an embodiment, it was confirmed that simply keeping the A8 and B10amino acids as their native threonine and histidine, respectively, doeseliminate the anti-drug antibody response, but the resulting insulin-Fcfusion protein is not bioactive in the target species (e.g., the NAOC isless than 150% FBGL·days·kg/mg). Therefore, it was necessary toexperiment with various A-chain, B-chain, and C-chain variations to finda suitable solution. Most variants failed to achieve homodimer titersgreater than 50 mg/L, and many of those that did meet those objectivesdid not reach acceptable levels of bioactivity in the target species(e.g., acceptable NAOC values of greater than 150% FBGL·days·kg/mg).Having screened over 120 variants, the following insulin polypeptide ofSEQ ID NO: 6_NULL was deemed suitable with respect to achievinghomodimer titers of greater than 50 mg/L, NAOC values in the targetspecies of greater than 150% FBGL·days·kg/mg, minimal immunogenicity,and NAOCR values after the third injection in the target species ofgreater than 0.5 of the associated insulin-Fc fusion proteins(non-native amino acids underlined and deleted native amino acidsrepresented with an underlined Z):

(SEQ ID NO: 6_NULL) FVNQHLCGSX₁ LVEALELVCGERGFHYZZZZGGGGGGSGGGGGIVEQCCX₂ STCSLDQLENYCX₃where X₁ is not D, X₂ is not H, and X₃ is absent or N.

In specific embodiments, in SEQ ID NO: 6_NULL, X₁ is H, X₂ is T, and X₃is absent or N resulting in the following SEQ ID NO: 7_NULL (withnon-native amino acids underlined and deleted native amino acidsrepresented with an underlined Z):

(SEQ ID NO: 7_NULL) FVNQHLCGSHLVEALELVCGERGFHYZZZZGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCX₃

where X₃ is absent or N.

In a specific embodiment, in SEQ ID NO: 7_NULL, X₃ is absent resultingin the following SEQ ID NO: 8_NULL (with non-native amino acidsunderlined and deleted native amino acids represented with an underlinedZ):

(SEQ ID NO: 8_NULL) FVNQHLCGSHLVEALELVCGERGFHYZZZZGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCZ

In a specific embodiment, in SEQ ID NO: 7_NULL, X₃ is N resulting in thefollowing SEQ ID NO: 9_NULL (with non-native amino acids underlined anddeleted native amino acids represented with an underlined Z):

(SEQ ID NO: 9_NULL) FVNQHLCGSHLVEALELVCGERGFHYZZZZGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCN

In some embodiments, the Fc fragment was mutated to preventglycosylation during synthesis and potentially reduce the immunogenicityof the resulting insulin-Fc fusion protein in the target animal (e.g.dog or cat). Unexpectedly, it was discovered that there was aninteraction between the insulin polypeptide and the mutated Fc fragmentsuch that yet another amino acid mutation was required on the insulinpolypeptide in order to render the insulin-Fc fusion proteinsufficiently manufacturable (e.g., with a homodimer titer greater than50 mg/L) and non-immunogenic with an NAOC value in the target species ofgreater than 150% FBGL·days·kg/mg and a NAOCR value after the thirdinjection in the target species of greater than 0.5. Specifically, itwas discovered that mutating the B16 amino acid to an alanine on theinsulin polypeptide was required when it was linked to specific,mutated, non-glycosylated Fc fragments resulting in the followinginsulin polypeptide SEQ ID NO: 10_NULL (with non-native amino acidsunderlined and deleted native amino acids represented with an underlinedZ):

(SEQ ID NO: 10_NULL) FVNQHLCGSX₁ LVEALALVCGERGFHYZZZZGGGGGGSGGGGGIVEQCCX₂ STCSLDQLENYCZwhere X₁ is not D and X₂ is not H.

In a specific embodiment, in SEQ ID NO: 10_NULL, X₁ is H and X₂ is Tresulting in the following SEQ ID NO: 11_NULL (with non-native aminoacids underlined and deleted native amino acids represented with anunderlined Z):

SEQ ID NO: 11_NULL FVNQHLCGSHLVEALALVCGERGFHYZZZZGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCZ -

The following are restatements of the sequences shown above but with theabsent amino acids of symbol Z removed from the notation of the insulinpolypeptide sequences. Again, in all cases the non-native amino acidsunderlined. To avoid confusion, each original sequence containing Zsymbols is listed above the new sequence with the Z symbols removed.Despite the two separate notations, the paired sequences refer toexactly the same insulin polypeptide.

SEQ ID NO: 6_NULL restated as:

(SEQ ID NO: 6) FVNQHLCGSX₁ LVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCX₂ STCSLDQLENYCX₃where X₁ is not D, X₂ is not H, and X₃ is absent or N.SEQ ID NO: 7_NULL restated as:

(SEQ ID NO: 7) FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCX₃where X₃ is absent or N.SEQ ID NO: 8_NULL restated as:

(SEQ ID NO: 8) FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCSEQ ID NO: 9_NULL restated as:

(SEQ ID NO: 9) FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCNSEQ ID NO: 10_NULL restated as:

(SEQ ID NO: 10) FVNQHLCGSX₁ LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCX₂ STCSLDQLENYCwhere X₁ is not D and X₂ is not H.SEQ ID NO: 11_NULL restated as:

(SEQ ID NO: 11) FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCLinker

The successful construction of a recombinantly made insulin-Fc fusionprotein requires a linker connecting the insulin polypeptide to the Fcfragment. In embodiments, an insulin-Fc fusion protein described hereincomprises a peptide linker between the insulin polypeptide and the Fcfragment comprising amino acids (e.g., natural or unnatural aminoacids). In embodiments, the peptide linker can be encoded by a nucleicacid molecule, for example such that a single nucleic acid molecule canencode the various peptides within an insulin polypeptide as well as thepeptide linker and the Fc fragment. The choice of peptide linker (forexample, the length, composition, hydrophobicity, and secondarystructure) could impact the manufacturability (i.e., the homodimertiter), the chemical and enzymatic stability, the bioactivity (i.e., theNAOC value), and the immunogenicity of the insulin-Fc fusion protein(Chen, X., Zaro, J., Shen, W. C., Adv Drug Deliv Rev. 2013 October 15;65(10): 1357-1369). Table 1 lists several linkers used in the design ofan insulin-Fc fusion protein with the goal of improving the homodimertiter and the bioactivity.

TABLE 1 Peptide Linker Between A-chain and FcFragment in an Insulin-Fc Fusion Protein GGGGAGGGG (SEQ ID NO: 12)GGGGSGGGG (SEQ ID NO: 13) GGGGGAGGGG (SEQ ID NO: 126)GGGGSGGGGSGGGGSGGGG (SEQ ID NO: 127)GGGGKGGGGKGGGGKGGGG (SEQ ID NO: 128)GGGGGQGGGGQGGGGQGGGGG (SEQ ID NO: 14)GGGGGAGGGGAGGGGAGGGGG (SEQ ID NO: 129)SGGGGQGGGGQGGGGQGGGGG (SEQ ID NO: 130)HGGGGQGGGGQGGGGQGGGGG (SEQ ID NO: 131)PGGGGGQGGGGQGGGGQGGGGG (SEQ ID NO: 132)In embodiments, the peptide linker comprises the sequence:

(SEQ ID NO: 12) GGGGAGGGG.In other embodiments, the peptide linker comprises the sequence:

(SEQ ID NO: 13) GGGGSGGGG.In preferred embodiments, the peptide linker comprises the sequence:

(SEQ ID NO: 14) GGGGGQGGGGQGGGGQGGGGG.

In constructing a recombinantly made insulin-Fc fusion protein with apeptide linker like the one of SEQ ID NO: 14, attention must be paid tothe possibility of unwanted enzymatic cleavage between the C-terminus ofthe insulin A-chain and the N-terminus of the peptide linker. Cleavageof the linker and Fc-fragment from the insulin polypeptide would renderthe insulin-Fc fusion protein incapable of providing an extendedduration of bioactivity. A known enzymatic cleavage site exits betweenasparagine-glycine bonds (Vlasak, J., Ionescu, R., (2011) MAbs Vol. 3,No. 3 pp 253-263). In many peptide linker embodiments, including thepreferred peptide linker of SEQ ID NO: 14, the N-terminal amino acid isa glycine. Furthermore, the C-terminus of the insulin A-chain i.e. (the21st amino acid from the N-terminus of the A-chain (i.e., A21)) is anasparagine. Therefore, the A21 asparagine is omitted in the insulinpolypeptides of SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 11 toeliminate the potentially enzymatically cleavable asparagine-glycinebond that would form between the A-chain and the peptide linker.Unexpectedly, an insulin-Fc fusion protein constructed from the insulinpolypeptide of SEQ ID NO: 9, which retains the asparagine at theC-terminus of the A-chain, demonstrates manufacturability in mammaliancells with an acceptable homodimer titer (i.e., a homodimer titergreater than 50 mg/L), an acceptable bioactivity in vivo (i.e., a NAOCgreater than 150% FBGL·days·kg/mg in the target animal), and sustainedlevels of bioactivity after multiple doses (i.e., a NAOCR values afterthe third injection in the target animal of greater than 0.5). Theresults indicate that, contrary to expectations based on priorteachings, there is no risk of enzymatic cleavage or deactivation ofinsulin-Fc fusion proteins containing the asparagine-glycine linkbetween the insulin polypeptide and peptide linker, at least forinsulin-Fc fusion proteins comprising the Fc fragment sequencesdisclosed herein.

Fc Fragment

The terms “Fc fragment,” “Fc region,” “Fc domain,” or “Fc polypeptide,”are used herein to define a C-terminal region of an immunoglobulin heavychain. The Fc fragment, region, domain or polypeptide may be a nativesequence Fc region or a variant/mutant Fc region. Although theboundaries of the Fc region of an immunoglobulin heavy chain may vary,they generally comprise some or all of the hinge region of the heavychain, the CH2 region of the heavy chain, and the CH3 region of theheavy chain. The hinge region of a canine or feline Fc fragmentcomprises amino acid sequences that connect the CH1 domain of the heavychain to the CH2 region of the heavy chain and contains one or morecysteines that form one or more interheavy chain disulfide bridges toform a homodimer of an Fc fusion protein from two identical but separatemonomers of the Fc fusion protein. The hinge region may comprise all orpart of a naturally occurring amino acid sequence or a non-naturallyoccurring amino acid sequence.

An Fc receptor (FcR) refers to a receptor that binds to an Fc fragmentor to the Fc region of an antibody. In embodiments, the FcR is a nativesequence of the canine or feline FcR. In embodiments, the FcR is onewhich binds an Fc fragment or the Fc region of an IgG antibody (a gammareceptor) and includes without limitation, receptors of the Fc(gamma)receptor I, Fc(gamma) receptor IIa, Fc(gamma) receptor IIb, andFc(gamma) receptor III subclasses, including allelic variants andalternatively spliced forms of these receptors. “FcR” also includes theneonatal receptor, FcRn, which is responsible for the transfer ofmaternal IgG molecules to the fetus (Guyer et al., 1976 J. Immunol.,117:587; and Kim et al., 1994, J. Immunol., 24:249) and is alsoresponsible for the prolonged in vivo elimination half-lives ofantibodies and Fc-fusion proteins in vivo. In embodiments, FcR of humanorigin are used in vitro (e.g., in an assay) to measure the binding ofinsulin-Fc fusion proteins comprising Fc fragments of canine or felineorigin so as to assess their FcR binding properties. Those skilled inthe art will understand that mammalian FcR from one species (e.g., FcRof human origin) are sometimes capable of in vitro binding of Fcfragments from a second species (e.g. FcR of canine or feline origin).In embodiments, FcR of canine origin are used in vitro (e.g., in anassay) to measure the binding of insulin-Fc fusion proteins comprisingFc fragments of both canine or feline origin so as to assess their FcRbinding properties. Those skilled in the art will understand thatmammalian FcR from one species (e.g., FcR of canine origin) are capableof in vitro binding of insulin-Fc fusion proteins comprising Fcfragments from the same species (e.g., of canine origin) and alsosometimes insulin-Fc fusion proteins comprising Fc fragments originatingfrom another mammalian species (e.g., of feline origin).

In embodiments, the Fc fragment comprises the Fc region (e.g., hingeregion, CH2 domain, and CH3 domain) of a mammalian IgG, for example acanine IgGA Fc fragment (SEQ ID NO: 15), a canine IgGB Fc fragment (SEQID NO: 16), a canine IgGC Fc fragment (SEQ ID NO: 17), or a canine IgGDFc fragment (SEQ ID NO: 18) or a feline IgG1a fragment (SEQ ID NO: 19),a feline IgG1b Fc fragment (SEQ ID NO: 20), or a feline IgG2 Fc fragment(SEQ ID NO: 21). In embodiments, the C-terminal lysine that is oftenfound in native canine or feline IgG isotype Fc fragment amino acidsequences (i.e., the lysine that represents the last amino acid of theFc fragment sequence) is omitted to prevent the accidental production ofunwanted amino acid sequence variants during manufacturing (e.g., Fcfragments containing the C-terminal lysine becoming mixed with Fcfragments where the C-terminal lysine is omitted, which can occur duringproduction of the desired protein within cells (Dick, L W., (2008)Biotechnol Bioeng. August 15; 100(6) pp 1132-43). Therefore, inembodiments, the canine and feline Fc fragment sequences lacking aC-terminal lysine are:

(SEQ ID NO: 15) RCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSPG (SEQ ID NO: 16)DCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 17)CNNCPCPGCGLLGGPSVFIFPPKPKDILVTARTPTVTCVVVDLDPENPEVQISWFVDSKQVQTANTQPREEQSNGTYRVVSVLPIGHQDWLSGKQFKCKVNNKALPSPIEEIISKTPGQAHQPNVYVLPPSRDEMSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEPESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQISLSHSPG (SEQ ID NO: 18)CISPCPVPESLGGPSVFIFPPKPKDILRITRTPEITCVVLDLGREDPEVQISWFVDGKEVHTAKTQPREQQFNSTYRVVSVLPIEHQDWLTGKEFKCRVNHIGLPSPIERTISKARGQAHQPSVYVLPPSPKELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEALQNHYTDLSLSHSPG (SEQ ID NO: 19)DCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKAKGQPHEPQVYVLPPAQEELSRNKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 20)DCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 21)GEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG

Replacing the human Fc with canine IgGA is preferable to minimize anyunwanted immunogenicity in dogs due to the IgGA isotype's lack ofFc(gamma) effector function in dogs (much like the human IgG2 isotype inhumans). However, in an embodiment containing the insulin polypeptide ofSEQ ID NO: 5 and the peptide linker of SEQ ID NO: 12, it wasunexpectedly discovered that the insulin-Fc fusion protein comprisingthe canine IgGA fragment (SEQ ID NO: 15) was highly aggregated with lowtiters of the desired homodimer (i.e., homodimer titers less than 50mg/L). Furthermore, the compound was non-bioactive in dogs (i.e., theNAOC value was less than 150% FBGL·days·kg/mg), presumably due to itshigh level of aggregation (e.g. low % homodimer). Despite mutating theinsulin polypeptide of SEQ ID NO: 5, the canine IgGA Fc fragment (SEQ IDNO: 15), and/or the linker, there was no embodiment based on the canineIgGA Fc fragment with a low enough degree of aggregation and a highenough titer of the desired homodimer. On the other hand, replacing ofthe canine IgGA Fc fragment (SEQ ID NO: 15) with the canine IgGB Fcfragment (SEQ ID NO: 16) yielded a much less aggregated compound with acomparatively high titer of the desired homodimer. Furthermore, thecompound containing the insulin polypeptide of SEQ ID NO: 5 and thecanine IgGB Fc fragment (SEQ ID NO: 16) was bioactive in dogs,exhibiting glucose lowering bioactivity over multiple days (i.e., theNAOC value was greater than 150% FBGL·days·kg/mg).

The preference for the canine IgGB Fc fragment over the canine IgGA Fcfragment was confirmed in embodiments containing the insulin polypeptideof SEQ ID NO: 8 and the peptide linker of SEQ ID NO: 14, both of whichvary considerably from the insulin polypeptide of SEQ ID NO: 5 and thepeptide linker of SEQ ID NO: 12. Insulin-Fc fusion proteins containingthe insulin polypeptide of SEQ ID NO: 8 and the peptide linker of SEQ IDNO: 14 were synthesized using Fc fragments from the canine IgGA (SEQ IDNO: 15), canine IgGB (SEQ ID NO: 16), canine IgGC (SEQ ID NO: 17), orcanine IgGD (SEQ ID NO: 18) immunoglobulins. Using the conventionalpurification method, only the compounds comprising the canine IgGA andthe canine IgGB showed any appreciable protein yields. However, justlike before, the canine IgGA version of the compound was highlyaggregated with low levels of bioactivity, whereas the canine IgGBversion of the compound exhibited a low degree of aggregation (i.e. high% homodimer), a high titer of the desired homodimer (i.e., a homodimertiter greater than 50 mg/L), and appreciable levels of long-durationglucose lowering bioactivity in dogs (i.e., the NAOC value was greaterthan 150% FBGL·days·kg/mg). Using an alternative purification method,the canine IgGC version of the compound was recovered with low degreesof aggregation, but it was minimally bioactive in dogs (i.e., the NAOCvalue was less than 150% FBGL·days·kg/mg), presumably due to its lowaffinity for the FcRn receptor. Therefore, with respect to adog-specific product, the canine IgGB (SEQ ID NO: 16) is the preferredFc fragment for all insulin-Fc fusion proteins used in dogs, regardlessof the choice of insulin polypeptide.

Replacing the human Fc with feline IgG2 is preferable to minimize anyunwanted immunogenicity in cats due to the IgG2 isotype's lack ofFc(gamma) effector function in cats (much like the human IgG2 isotype inhumans). Unlike the case with the dogs, in embodiments containing theinsulin polypeptide of SEQ ID NO: 4, it was discovered that insulin-Fcfusion proteins comprising the feline IgG2 fragment (SEQ ID NO: 21) andthe feline IgG1b fragment (SEQ ID NO: 20) were similarly high yieldingwith low degrees of aggregation (i.e., homodimer titers greater than 50mg/L) and appreciable insulin receptor affinity (i.e., insulin receptorIC50 values less than 5000 nM). However, unexpectedly when the insulinpolypeptide was changed to SEQ ID NO: 7, the insulin-Fc fusion proteincomprising the feline IgG2 fragment (SEQ ID NO: 21) was not bioactive incats (i.e., the NAOC was less than 150% FBGL·days·kg/mg), whereas theinsulin-Fc fusion protein comprising the feline IgG1b fragment (SEQ IDNO: 20) exhibited a low degree of aggregation (i.e., high % homodimer),a high titer of the desired homodimer (i.e., a homodimer titer greaterthan 50 mg/L), and appreciable levels of long-duration glucose loweringbioactivity in cats (i.e., the NAOC value was greater than 150%FBGL·days·kg/mg). Therefore, with respect to a cat-specific product, thefeline IgG1b fragment (SEQ ID NO: 20) is the preferred Fc fragment whenthe insulin polypeptide sequence comprises SEQ ID NO: 7.

Given that the canine IgGB and feline IgG1b isotypes interact with theirrespective species-specific Fc(gamma) receptors with higher affinitiesthan their canine IgGA and feline IgG2 isotype counterparts, there mayor may not be a risk of unwanted immunogenicity after repeatedinjections. One method for reducing the Fc(gamma) interaction involvesdeglycosylating or preventing the glycosylation of the Fc fragmentduring synthesis in the host cell. Each IgG fragment contains aconserved asparagine (N)-glycosylation site in the CH2 domain of eachheavy chain of the Fc region. Herein, the notation used to refer to theconserved N-glycosylation site is “cNg”. One way to remove the attachedglycan from a synthesized insulin-Fc fusion protein is to mutate the cNgsite to prevent the attachment of glycans altogether during productionin the host cell. Herein, the notation used to describe a cNg mutationis cNg-(substituted amino acid). For example, if the asparagine at thecNg site is mutated to serine, this mutation is notated as “cNg-S”.

The absolute position of the cNg site from the N-terminus of the B-chainof the insulin-Fc fusion protein varies depending on the length of theinsulin polypeptide, the length of the linker, and any omitted aminoacids in the Fc fragment prior to the cNg site. Herein, the notationused to refer to the absolute position of the cNg site in a giveninsulin-Fc fusion protein sequence (as measured counting from theN-terminus of the B-chain of the insulin-Fc fusion protein) is“NB(number)”. For example, if the cNg site is found at the 151^(st)amino acid position as counted from the N-terminus of the B-chain, theabsolute position of this site is referred to as cNg-NB151. As a furtherexample, if the cNg site is found at the 150 amino acid position ascounted from the N-terminus of the B-chain, and the asparagine at thissite is mutated to serine, this mutation is noted as “cNg-NB151-S”.

In embodiments containing the insulin polypeptide of SEQ ID NO: 5 andthe canine IgGB Fc fragment with the cNg-Q, cNg-S, cNg-D, and cNg-Kmutations, it was unexpectedly discovered that only the compoundscontaining the cNg-K and cNg-S mutations exhibited the requisitehomodimer titer greater than 50 mg/L and lowest Fc(gamma)RI bindingaffinities. On the other hand, in an embodiment containing the insulinpolypeptide of SEQ ID NO: 8 and the canine IgGB Fc fragment with thecNg-S mutation, it was unexpectedly discovered that the resultingcompound was significantly less bioactive in dogs compared to the nativecanine IgGB Fc-containing counterpart (i.e., the NAOC value wassignificantly lower for the counterpart containing the nativeglycosylation site amino acid, e.g., cNg-N). The bioactivity wasunexpectedly restored in the cNg-S mutant (i.e., the NAOC valueincreased significantly) when the B16 amino acid was mutated to alanineas described above for insulin polypeptide SEQ ID NO: 11. Takentogether, there is an unexpected and significant interaction between thechoice of cNg mutation and the composition of the insulin polypeptidesuch that experimentation is required to identify the preferredembodiments. In specific embodiments, the canine IgGB Fc mutantcontaining the cNg-S mutation is preferred and the sequence withunderlined cNg-S is shown as:

(SEQ ID NO: 22) DCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGIn specific embodiments, the feline IgG1b Fc mutant containing the cNg-Smutation is preferred:

(SEQ ID NO: 23) DCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFSSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCS VSHEALHSHHTQKSLTQSPGInsulin-Fc Fusion Proteins

Provided herein are insulin-Fc fusion proteins comprising an insulinpolypeptide, an Fc fragment, and a linker between the insulinpolypeptide and the Fc fragment. In embodiments, the insulin polypeptidecomprises domains in the following orientation from N- toC-termini--(N-terminus)--B-chain--C-chain--A-chain--(C-terminus). Inembodiments, the insulin polypeptide is located on the N-terminal sideof the Fc fragment. In embodiments, the fusion protein comprises domainsin the following orientation from N- to C-termini--(N-terminus)--insulinpolypeptide--linker--Fc fragment--(C-terminus) (e.g.,(N-terminus)--B-chain--C-chain--A-chain--linker--Fcfragment--(C-terminus)) as illustrated in FIG. 1.

In preferred embodiments, the preferred non-immunogenic, bioactiveinsulin polypeptide of SEQ ID NO: 6 is combined with the preferredcanine IgGB Fc fragment of SEQ ID NO: 16 using the preferred linker ofSEQ ID NO: 14 to produce a family of high homodimer titer-yielding,non-aggregated, bioactive, non-immunogenic insulin-Fc fusion proteins ofSEQ ID NO: 24 that exhibit homodimer titers greater than 50 mg/L, NAOCvalues greater than 150% FBGL·days·kg/mg in dogs, and NAOCR valuesgreater than 0.5 after the third injection in a series of repeatedinjections in dogs. The following shows SEQ ID NO: 24 with non-nativeamino acids underlined:

(SEQ ID NO: 24) FVNQHLCGSX₁ LVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCX₂ STCSLDQLENYCX₃ GGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGwhere X₁ is not D, X₂ is not H, and X₃ is absent or N.

In preferred embodiments comprising SEQ ID NO: 24, the X₁ is H, X₂ is T,and X₃ is absent or N. The selections produce the high homodimertiter-yielding, non-aggregated, bioactive, non-immunogenic insulin-Fcfusion proteins of SEQ ID NO: 25 that exhibit homodimer titers greaterthan 50 mg/L, NAOC values greater than 150% FBGL·days·kg/mg in dogs, andNAOCR values greater than 0.5 after the third injection in a series ofrepeated injections in dogs. The following shows SEQ ID NO: 25 withnon-native amino acids underlined:

(SEQ ID NO: 25) FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCX₃ GGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGwhere X₃ is absent or N.

In preferred embodiments, X₃ is absent in SEQ ID NO: 25 to produce thehigh homodimer-yielding, non-aggregated, bioactive, non-immunogenicinsulin-Fc fusion protein of SEQ ID NO: 32 that exhibits a homodimertiter greater than 50 mg/L, a NAOC value greater than 150%FBGL·days·kg/mg in dogs, and a NAOCR value greater than 0.5 after thethird injection in a series of repeated injections in dogs. Thefollowing shows SEQ ID NO: 32 with non-native amino acids underlined:

(SEQ ID NO: 32) FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

In preferred embodiments, X₃ is N in SEQ ID NO: 25 to produce the highhomodimer titer-yielding, non-aggregated, bioactive, non-immunogenicinsulin-Fc fusion protein of SEQ ID NO: 34 that exhibits a homodimertiter greater than 50 mg/L, a NAOC value greater than 150%FBGL·days·kg/mg in dogs, and a NAOCR value greater than 0.5 after thethird injection in a series of repeated injections in dogs. Thefollowing shows SEQ ID NO: 34 with non-native amino acids underlined:

(SEQ ID NO: 34) FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCNGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

In preferred embodiments, the preferred non-glycosylated, cNg-S mutatedcanine IgGB Fc fragment of SEQ ID NO: 22 is combined with the preferredB16A mutated insulin polypeptide sequence of SEQ ID NO: 10 using thepreferred linker of SEQ ID NO: 14 to produce a family of high homodimertiter-yielding, non-aggregated, bioactive, non-immunogenic insulin-Fcfusion proteins of SEQ ID NO: 26 that exhibit homodimer titers greaterthan 50 mg/L, NAOC values greater than 150% FBGL·days·kg/mg in dogs, andNAOCR values greater than 0.5 after the third injection in a series ofrepeated injections in dogs. The following shows SEQ ID NO: 26 withnon-native amino acids underlined:

(SEQ ID NO: 26) FVNQHLCGSX₁ LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCX₂ STCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGwhere X₁ is not D and X₂ is not H.

In a preferred embodiment, the X₁ is H and X₂ is T in SEQ ID NO: 26 toproduce the high homodimer titer-yielding, non-aggregated, bioactive,non-immunogenic insulin-Fc fusion protein of SEQ ID NO: 36 that exhibitsa homodimer titer greater than 50 mg/L, a NAOC value greater than 150%FBGL·days·kg/mg in dogs, and a NAOCR value greater than 0.5 after thethird injection in a series of repeated injections in dogs. Thefollowing shows SEQ ID NO: 36 with non-native amino acids underlined:

(SEQ ID NO: 36) FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

In preferred embodiments, the preferred non-immunogenic, bioactiveinsulin polypeptide of SEQ ID NO: 6 where X₃ is absent is combined withthe preferred feline IgG1b Fc fragment of SEQ ID NO: 20 using thepreferred linker of SEQ ID NO: 14 to produce a family of high homodimertiter-yielding, non-aggregated, bioactive, non-immunogenic insulin-Fcfusion proteins of SEQ ID NO: 27 that exhibit homodimer titers greaterthan 50 mg/L, NAOC values greater than 150% FBGL·days·kg/mg in cats, andNAOCR values greater than 0.5 after the third injection in a series ofrepeated injections in cats. The following shows SEQ ID NO: 27 withnon-native amino acids underlined:

(SEQ ID NO: 27) FVNQHLCGSX₁ LVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCX₂ STCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPGwhere X₁ is not D and X₂ is not H.

In a preferred embodiment, the X1 is H and X2 is T in SEQ ID NO: 27 toproduce the high homodimer titer-yielding, non-aggregated, bioactive,non-immunogenic insulin-Fc fusion protein of SEQ ID NO: 38 that exhibitsa homodimer titer greater than 50 mg/L, a NAOC value greater than 150%FBGL·days·kg/mg in cats, and a NAOCR value greater than 0.5 after thethird injection in a series of repeated injections in cats. Thefollowing shows SEQ ID NO: 38 with non-native amino acids underlined:

(SEQ ID NO: 38) FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG

In preferred embodiments, the preferred non-glycosylated, cNg-S mutatedfeline IgG1b Fc fragment of SEQ ID NO: 23 is combined with the preferredB16A mutated insulin polypeptide sequence of SEQ ID NO: 10 using thepreferred linker of SEQ ID NO: 14 to produce a family of high homodimertiter-yielding, non-aggregated, bioactive, non-immunogenic insulin-Fcfusion proteins of SEQ ID NO: 28 that exhibit homodimer titers greaterthan 50 mg/L, NAOC values greater than 150% FBGL·days·kg/mg in cats, andNAOCR values greater than 0.5 after the third injection in a series ofrepeated injections in cats. The following shows SEQ ID NO: 28 withnon-native amino acids underlined:

(SEQ ID NO: 28) FVNQHLCGSX₁ LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCX₂ STCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFSSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPGwhere X₁ is not D and X₂ is not H.

In a preferred embodiment, the X₁ is H and X₂ is T in SEQ ID NO: 28 toproduce the high homodimer titer-yielding, non-aggregated, bioactive,non-immunogenic insulin-Fc fusion protein of SEQ ID NO: 40 that exhibitsa homodimer titer greater than 50 mg/L, a NAOC value greater than 150%FBGL·days·kg/mg in cats, and a NAOCR value greater than 0.5 after thethird injection in a series of repeated injections in dogs. Thefollowing shows SEQ ID NO: 40 with non-native amino acids underlined:

(SEQ ID NO: 40) FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFSSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG

In some embodiments, an insulin-Fc fusion protein described herein doesnot include a leader amino acid sequence at the N-terminus. In otherembodiments, an insulin-Fc fusion protein described herein includes aleader sequence, e.g., at the N-terminus. An exemplary leader sequenceincludes the amino acid sequence MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 30). Insome embodiments, an insulin-Fc fusion protein described herein isencoded by a nucleic acid molecule comprising a leader sequence, e.g.,for expression (e.g., recombinant expression) in cells (e.g.,eukaryotic, e.g., mammalian cells). In certain embodiments, the leadersequence is cleaved off, e.g., in the cell culture, during expression.An exemplary nucleic acid sequence encoding a leader sequence includesthe nucleic acid sequence:

(SEQ ID NO: 29) atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactcc.

Also disclosed herein are nucleic acid sequences (e.g., cDNA) encodingthe insulin-Fc fusion proteins of SEQ ID NOs: 032, 034, 036, 038, and040.

In the embodiment comprising the insulin-Fc fusion protein of SEQ ID NO:32, the nucleic acid sequence (leader sequence underlined) is:

(SEQ ID NO: 31) atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggaactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaagtgccccgctcccgagatgctgggcggacccagcgtgttcatcttccctcccaagcccaaggacacactgctgatcgccaggaccccggaggtgacctgcgtggtggtggacctggatcccgaagaccccgaggtgcagatcagctggttcgtggatggaaagcagatgcagaccgccaagacccaaccccgggaagagcagttcaacggcacctacagggtggtgagtgtgttgcccatcggccaccaggactggctgaaggggaagcaattcacatgcaaggttaataacaaggccctgcccagccccatcgagaggaccatcagcaaggccaggggccaggcccaccagccatctgtgtacgtgctgcccccatctagggaggaactgagcaagaacacagtcagccttacttgcctgatcaaggacttcttcccaccggacatagacgtggagtggcagagtaacggccagcaggagcccgagagcaagtataggaccacaccgccccaactggacgaggacggaagctacttcctctacagcaaattgagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgcgccgtgatgcacgaggctttgcataaccactacacccaggagagcctgtcccacagccccggatag

In the embodiment comprising the insulin-Fc fusion protein of SEQ ID NO:34, the nucleic acid sequence (leader sequence underlined) is:

(SEQ ID NO: 33) atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggaactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcaacggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaagtgccccgctcccgagatgctgggcggacccagcgtgttcatcttccctcccaagcccaaggacacactgctgatcgccaggaccccggaggtgacctgcgtggtggtggacctggatcccgaagaccccgaggtgcagatcagctggttcgtggatggaaagcagatgcagaccgccaagacccaaccccgggaagagcagttcaacggcacctacagggtggtgagtgtgttgcccatcggccaccaggactggctgaaggggaagcaattcacatgcaaggttaataacaaggccctgcccagccccatcgagaggaccatcagcaaggccaggggccaggcccaccagccatctgtgtacgtgctgcccccatctagggaggaactgagcaagaacacagtcagccttacttgcctgatcaaggacttcttcccaccggacatagacgtggagtggcagagtaacggccagcaggagcccgagagcaagtataggaccacaccgccccaactggacgaggacggaagctacttcctctacagcaaattgagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgcgccgtgatgcacgaggctttgcataaccactacacccaggagagcctgtcccacagccccggatag

In the embodiment comprising the insulin-Fc fusion protein of SEQ ID NO:36, the nucleic acid sequence (leader sequence underlined) is:

(SEQ ID NO: 35) atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggcactcgtgtgcggcgagcggggcttccactacgggggtggcggaggaggttctggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaagtgccccgctcccgagatgctgggcggacccagcgtgttcatcttccctcccaagcccaaggacacactgctgatcgccaggaccccggaggtgacctgcgtggtggtggacctggatcccgaagaccccgaggtgcagatcagctggttcgtggatggaaagcagatgcagaccgccaagacccaaccccgggaagagcagttctcaggcacctacagggtggtgagtgtgttgcccatcggccaccaggactggctgaaggggaagcaattcacatgcaaggttaataacaaggccctgcccagccccatcgagaggaccatcagcaaggccaggggccaggcccaccagccatctgtgtacgtgctgcccccatctagggaggaactgagcaagaacacagtcagccttacttgcctgatcaaggacttcttcccaccggacatagacgtggagtggcagagtaacggccagcaggagcccgagagcaagtataggaccacaccgccccaactggacgaggacggaagctacttcctctacagcaaattgagcgttgacaaaagcaggtggcagcgaggcgacaccttcatctgcgccgtgatgcacgaggctttgcataaccactacacccaggagagcctgtcccacagccccggatag

In the embodiment comprising the insulin-Fc fusion protein of SEQ ID NO:38, the nucleic acid sequence (leader sequence underlined) is:

(SEQ ID NO: 37) atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggaactcgtgtgcggcgagcggggcaccactacgggggtggcggaggaggactggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaaatgtcctccgcctgagatgctgggtggccctagcatatcatcacccgcccaagcccaaggatactctgtccattagcaggacccccgaggtgacctgcctggtggtggacctggggccagacgactctgacgtgcagatcacctggacgtagacaacacccaggatacactgccaagaccagtcccagggaggagcagacaacagcacatacagggtggtgagcgactgcccatcctgcaccaggactggctgaaaggcaaagagttcaagtgtaaggtgaacagcaagagcctgcccagccccattgaaaggaccatcagcaaggacaagggccagccgcacgagccccaagtctacgtgctgcccccagcacaggaagagctgagcaggaacaaggttagcgtgacatgcctgatcgagggtactaccccagcgacatcgccgtggagtgggaaatcaccggccaacccgagcccgagaacaactacaggaccactccgccgcaactggacagcgacgggacctacttcagtatagcaggctgagcgtggaccggagcaggtggcagaggggcaacacctacacttgcagcgtgagccacgaggccagcacagccaccacactcagaagagtctgacccagagcccgggatag

In the embodiment comprising the insulin-Fc fusion protein of SEQ ID NO:40, the nucleic acid sequence (leader sequence underlined) is:

(SEQ ID NO: 39) atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactccttcgtgaaccagcacctgtgcggctcccacctggtggaagctctggcactcgtgtgcggcgagcggggcaccactacgggggtggcggaggaggactggtggcggcggaggcatcgtggaacagtgctgcacctccacctgctccctggaccagctggaaaactactgcggtggcggaggtggtcaaggaggcggtggacagggtggaggtgggcagggaggaggcgggggagactgccccaaatgtcctccgcctgagatgctgggtggccctagcatatcatcacccgcccaagcccaaggatactctgtccattagcaggacccccgaggtgacctgcctggtggtggacctggggccagacgactctgacgtgcagatcacctggacgtagacaacacccaggatacactgccaagaccagtcccagggaggagcagttcagcagcacatacagggtggtgagcgttctgcccatcctgcaccaggactggctgaaaggcaaagagttcaagtgtaaggtgaacagcaagagcctgcccagccccattgaaaggaccatcagcaaggacaagggccagccgcacgagccccaagtctacgtgctgcccccagcacaggaagagctgagcaggaacaaggttagcgtgacatgcctgatcgagggtactaccccagcgacatcgccgtggagtgggaaatcaccggccaacccgagcccgagaacaactacaggaccactccgccgcaactggacagcgacgggacctacttcagtatagcaggctgagcgtggaccggagcaggtggcagaggggcaacacctacacttgcagcgtgagccacgaggccagcacagccaccacactcagaagagtctgacccagagcccgggatagInsulin-Fc fusion Protein Production

In embodiments, a fusion protein can be expressed by a cell as describedin more detail in the Examples section.

Expression and Purification

In embodiments, an insulin-Fc fusion protein can be expressedrecombinantly, e.g., in a eukaryotic cell, e.g., mammalian cell ornon-mammalian cell. Exemplary mammalian cells used for expressioninclude HEK cells (e.g., HEK293 cells) or CHO cells. CHO cells can besubdivided into various strains or subclasses, (e.g. CHO DG44, CHO-M,and CHO-K1), and some of these cell strains may be geneticallyengineered for optimal use with a particular type of nucleic acidmolecule (e.g., a vector comprising DNA) or a particular cell growthmedia composition as described in the Examples section. In embodiments,cells are transfected with a nucleic acid molecule (e.g., vector)encoding the insulin-Fc fusion protein (e.g., where the entireinsulin-Fc fusion protein is encoded by a single nucleic acid molecule).In embodiments, HEK293 cells are transfected with a vector that encodesfor the insulin-Fc fusion protein, but only results in temporaryexpression of the insulin-Fc fusion protein for a period of time (e.g.,3 days, 4 days, 5, days, 7 days, 10 days, 12 days, 14 days, or more)before the host cell stops expressing appreciable levels of theinsulin-Fc fusion protein (i.e., transient transfection). HEK293 cellsthat are transiently transfected with nucleic acid sequences encodingfor insulin-Fc fusion proteins often allow for more rapid production ofrecombinant proteins which facilitates making and screening multipleinsulin-Fc fusion protein candidates. In embodiments, CHO cells aretransfected with a vector that is permanently incorporated into the hostcell DNA and leads to consistent and permanent expression (i.e., stabletransfection) of the insulin-Fc fusion protein as long as the cells arecultured appropriately. CHO cells and CHO cell lines that are stablytransfected with nucleic acids encoding for insulin-Fc fusion proteinsoften take longer to develop, but they often produce higher proteinyields and are more amenable to manufacturing low cost products (e.g.,products for use in the veterinary pharmaceutical market). Cells andcell lines can be cultured using standard methods in the art. Inpreferred embodiments, HEK cells comprising any one of the cDNAsequences with SEQ ID NOs: 31, 33, 35, 37, and 39 are used to expressinsulin-Fc fusion proteins. In preferred embodiments, CHO cellscomprising any one of the cDNA sequences with SEQ ID NOs: 31, 33, 35,37, and 39 are used to express insulin-Fc fusion proteins.

In some embodiments, the insulin-Fc fusion protein is purified orisolated from the cells (e.g., by lysis of the cells). In otherembodiments, the insulin-Fc fusion protein is secreted by the cells andpurified or isolated from the cell culture media in which the cells weregrown. Purification of the insulin-Fc fusion protein can include usingcolumn chromatography (e.g., affinity chromatography) or using otherseparation methods based on differences in size, charge, and/or affinityfor certain molecules. In embodiments, purification of the insulin-Fcfusion protein involves selecting or enriching for proteins containingan Fc fragment, e.g., by using Protein A beads or a Protein A columnthat cause proteins containing an Fc fragment to become bound with highaffinity at neutral solution pH to the Protein A covalently conjugatedto the Protein A beads. The bound insulin-Fc fusion protein may then beeluted from the Protein A beads by a change in a solution variable (e.g.a decrease in the solution pH). Other separation methods such as ionexchange chromatography and/or gel filtration chromatography can also beemployed alternatively or additionally. In embodiments, purification ofthe insulin-Fc fusion protein further comprises filtering orcentrifuging the protein preparation. In embodiments, furtherpurification of the insulin-Fc fusion protein comprises dialfiltration,ultrafiltration, and filtration through porous membranes of varioussizes, as well as final formulation with excipients.

The purified insulin-Fc fusion protein can be characterized, e.g., forpurity, protein yield, structure, and/or activity, using a variety ofmethods, e.g., absorbance at 280 nm (e.g., to determine protein yield),size exclusion or capillary electrophoresis (e.g., to determine themolecular weight, percent aggregation, and/or purity), mass spectrometry(MS) and/or liquid chromatography (LC-MS) (e.g., to determine purityand/or glycosylation), and/or ELISA (e.g., to determine extent ofbinding, e.g., affinity, to an anti-insulin antibody). Exemplary methodsof characterization are also described in the Examples section.

In embodiments, the protein yield of an insulin-Fc fusion protein afterproduction in transiently transfected HEK cells and protein Apurification is greater than 5 mg/L, 10 mg/L, or 20 mg/L. In preferredembodiments, the protein yield of an insulin-Fc fusion protein afterproduction in transiently transfected HEK cells and protein Apurification is greater than 50 mg/L (e.g., greater than 60 mg/L,greater than 70 mg/L, greater than 80 mg/L, greater than 90 mg/L,greater than 100 mg/L). In embodiments, the % homodimer of an insulin-Fcfusion protein after production in transiently transfected HEK cells andprotein A purification is greater than 70% (e.g., greater than 80%,greater than 85%, greater than 90%, greater than 95%, greater than 96%,greater than 97%, greater than 98%, greater than 99%). In embodiments,the homodimer titer of an insulin-Fc fusion protein after production intransiently transfected HEK cells and protein A purification, calculatedas the product between the insulin-Fc fusion protein yield and the %homodimer is greater than 50 mg/L (e.g., greater than 60 mg/L, greaterthan 70 mg/L, greater than 80 mg/L, greater than 90 mg/L, greater than100 mg/L). Only candidates with a homodimer titer of greater than 50mg/L were considered useful in the present invention, because experiencehas demonstrated that homodimer titers less than this level will notlikely result in commercial production titers in CHO cells that meet thestringently low manufacturing cost requirements for veterinary products.

In embodiments, the protein yield of an insulin-Fc fusion protein afterproduction in stably transfected CHO cells (e.g., CHO cell lines or CHOcell clones) and protein A purification is greater than 100 mg ofinsulin-Fc fusion protein per L (e.g. mg/L of culture media). Inpreferred embodiments, the protein yield of an insulin-Fc fusion proteinafter production in stably transfected CHO cells (e.g. CHO cell lines orCHO cell clones) and protein A purification is greater than 150 mginsulin-Fc fusion protein/L of culture media (e.g., greater than 200mg/L, greater than 300 mg/L, greater than 400 mg/L, greater than 500mg/L, greater than 600 mg/L or more). In embodiments, the % homodimer ofan insulin-Fc fusion protein after production in stably transfected CHOcells (e.g. CHO cell lines or CHO cell clones) and protein Apurification is greater than 70% (e.g., greater than 80%, greater than85%, greater than 90%, greater than 95%, greater than 96%, greater than97%, greater than 98%, greater than 99%). In embodiments, the homodimertiter of an insulin-Fc fusion protein after production in stablytransfected CHO cells (e.g. CHO cell lines or CHO cell clones) andprotein A purification, calculated as the product between the insulin-Fcfusion protein yield and the % homodimer is greater than 150 mg/L (e.g.,greater than 200 mg/L, greater than 300 mg/L, greater than 400 mg/L,greater than 500 mg/L, greater than 600 mg/L or more).

Functional Features of Insulin-Fc Fusion Proteins

Described herein are methods for interacting with the insulin receptorsto lower blood glucose in companion animals (e.g., dogs or cats),wherein the method comprises administering to a subject an insulin-Fcfusion protein, e.g., a fusion protein described herein. In someembodiments, the subject has been diagnosed with diabetes (e.g., caninediabetes or feline diabetes).

In embodiments, an insulin-Fc fusion protein described herein binds tothe insulin receptor with an appreciable affinity as measured by theIC50 in the 4° C. IM-9 insulin receptor binding assay described inExample 7 (e.g. IC50 less than 5000 nM, IC50 less than 4000 nM, IC50less than 3000 nM, IC50 less than 2500 nM). Based on experience, onlycompounds exhibiting insulin receptor activity IC50 values less than5000 nM were deemed likely to exhibit bioactivity in the target species.Generally, higher affinity insulin receptor binding (i.e., lower IC50values) is preferred. However, it is well-known that the clearance ofinsulin and insulin analogs (e.g., insulin polypeptides describedherein) is governed primarily through binding to the insulin receptorfollowed by insulin receptor internalization and degradation within thecell. Therefore, insulin-Fc fusion proteins with too high of an insulinreceptor binding affinity (i.e., too low of an IC50) may be cleared tooquickly from circulation resulting in a lower than desired duration ofglucose-lowering bioactivity in the target animal.

In embodiments, an insulin-Fc fusion protein described herein is capableof lowering glucose levels (e.g., blood glucose levels) afteradministration in a subject. In embodiments, the glucose loweringactivity of the insulin-Fc fusion protein is greater than that of aninsulin reference standard. In some embodiments, the duration ofactivity of the insulin-Fc fusion protein can be measured by a decrease,e.g., a statistically significant decrease, in fasting blood glucoserelative to a pre-dose fasting blood glucose level. In embodiments, theduration of activity of the insulin-Fc fusion protein (e.g., the timeduring which there is a statistically significant decrease in fastingblood glucose level in a subject relative to a pre-dose level) is longerthan about 2 hours. In embodiments, the duration of activity of theinsulin-Fc fusion protein (e.g., the time during which there is astatistically significant decrease in blood glucose level in a subjectrelative to a pre-dose level) is longer than about 6 hours, 9 hours, 12hours, 18 hours, 1 day, 1.5 days, 2 days, 2.5 days, 3 days, 4 days, 5days, 6 days, 7 days, or longer. In embodiments, the insulin-Fc fusionprotein is long-acting (e.g., has a long half-life, e.g., in serum).

In embodiments, the serum half-life of the insulin-Fc fusion protein inthe target animal (e.g., dog or cat) is longer than that of an insulinreference standard or control formulation. In embodiments, the serumhalf-life of the insulin-Fc fusion protein (e.g., in the blood of asubject upon administration) in the target animal (e.g., dog or cat) islonger than about 2 hours. In embodiments, the serum half-life of theinsulin-Fc fusion protein in the target animal (e.g., dog or cat) isabout 0.5 days, 1 day, 2 days, or 2.5 days. In preferred embodiments,the serum half-life of the insulin-Fc fusion protein in the targetanimal (e.g., dog or cat) is about 3 days or longer.

In embodiments, the combination of potency and duration of bioactivitymay be quantified by calculating the area over the percent fasting bloodglucose (% FBGL) curve normalized to a given dose in mg/kg (NAOC) withunits of % FBGL·days·kg/mg. In embodiments, the NAOC of the insulin-Fcfusion protein is greater than 150% FBGL·days·kg/mg (e.g. greater than200% FBGL·days·kg/mg, greater than 250% FBGL·days·kg/mg or more). Again,based on experience, at NAOC values greater than 150% FBGL·days·kg/mg,the dose requirements in the target species will be sufficiently low soas to achieve an acceptable treatment cost. In embodiments, the NAOC ofthe insulin-Fc fusion protein must be maintained after repeated dosingin the target species (i.e., the ratio of the NAOC after the third doseto the NAOC after the first dose of the insulin-Fc fusion protein isgreater than 0.50 (e.g., greater than 0.60, greater than 0.70. greaterthan 0.80, greater than 0.90, or more).

In some embodiments, the insulin-Fc fusion protein described hereinbinds to the Fc(gamma) receptor with an affinity that is lower than thatof an insulin-Fc fusion protein reference standard as measured accordingto Example 8. In some embodiments, the ratio of the Fc(gamma) receptoraffinity of the insulin-Fc fusion protein to that of an insulin-Fcfusion protein reference standard is less than 0.50 (e.g. less than0.40, less than 0.30, less than 0.20).

Methods of Treatment and Characteristics of Subject Selection

Described herein are methods for treating diabetes (e.g., caninediabetes or feline diabetes), the methods comprising the administrationof an insulin-Fc fusion protein (e.g., an insulin-Fc fusion proteindescribed herein) to a subject.

In embodiments, a reference standard used in any method described hereincomprises a reference treatment or reference therapy. In someembodiments, the reference comprises a standard of care agent fordiabetes treatment (e.g., a standard of care agent for canine diabetesor a standard of care agent for feline diabetes). In some embodiments,the reference standard is a commercially available insulin or insulinanalog. In some embodiments, the reference standard comprises along-lasting insulin, intermediate-lasting insulin, short-lastinginsulin, rapid-acting insulin, short-acting, intermediate-acting,long-acting insulin. In some embodiments, the reference standardcomprises Vetsulin®, Prozinc®, insulin NPH, insulin glargine (Lantus®),or recombinant human insulin.

In embodiments, a reference standard used in any method described hereinincludes an outcome, e.g., outcome described herein, of a diabetestherapy (e.g., a canine diabetes therapy or a feline diabetes therapy).

In embodiments, a reference standard is a level of a marker (e.g., bloodglucose or fructosamine) in the subject prior to initiation of atherapy, e.g., an insulin-Fc fusion protein therapy described herein;where the subject has diabetes. In embodiments, the blood glucose levelin a companion animal (e.g. dog or cat) is greater than 200 mg/dL (e.g.greater than 250 mg/dL, 300 mg/dL, 350 mg/dL, 400 mg/dL or more) priorto initiation of therapy. In embodiments, the fructosamine level in acompanion animal (e.g. dog or cat) is greater than 250 micromol/L, 350micromol/L (e.g. greater than 400 micromol/L, 450 micromol/L, 500micromol/L, 550 micromol/L, 600 micromol/L, 650 micromol/L, 700micromol/L, 750 micromol/L or more) prior to initiation of therapy. Inembodiments, a reference standard is a measure of the presence of or theprogression of or the severity of the disease. In embodiments, areference standard is a measure of the presence of or the severity ofthe disease symptoms prior to initiation of a therapy, e.g., aninsulin-Fc fusion protein therapy described herein, e.g., where thesubject has diabetes.

Pharmaceutical Compositions and Routes of Administration

Provided herein are pharmaceutical compositions containing an insulin-Fcfusion protein described herein that can be used to lower blood glucosein companion animals (e.g. dogs or cats). The amount and concentrationof the insulin-Fc fusion protein in the pharmaceutical compositions, aswell as the quantity of the pharmaceutical composition administered to asubject, can be selected based on clinically relevant factors, such asmedically relevant characteristics of the subject (e.g., age, weight,gender, other medical conditions, and the like), the solubility ofcompounds in the pharmaceutical compositions, the potency and activityof the compounds, and the manner of administration of the pharmaceuticalcompositions. For further information on Routes of Administration andDosage Regimes the reader is referred to Chapter 25.3 in Volume 5 ofComprehensive Medicinal Chemistry (Corwin Hansch; Chairman of EditorialBoard), Pergamon Press 1990.

Formulations of the present disclosure include those suitable forparenteral administration. The phrases “parenteral administration” and“administered parenterally” as used herein means modes of administrationother than enteral and topical administration, usually by intravenous orsubcutaneous injection.

Examples of suitable aqueous and non-aqueous carriers that may beemployed in the pharmaceutical compositions of the disclosure includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants, e.g., Tween-like surfactants. In some embodiments, thepharmaceutical composition (e.g., as described herein) comprises aTween-like surfactant, e.g., polysorbate-20, Tween-20 or Tween-80. Insome embodiments, the pharmaceutical composition (e.g., as describedherein) comprises a Tween-like surfactant, e.g., Tween-80, at aconcentration between about 0.001% and about 2%, or between about 0.005%and about 0.1%, or between about 0.01% and about 0.5%.

In some embodiments, the concentration of the insulin-Fc fusion proteinin the aqueous carrier is about 3 mg/mL. In some embodiments, theconcentration of the insulin-Fc fusion protein in the aqueous carrier isabout 6 mg/mL. In some embodiments, the concentration of the insulin-Fcfusion protein in the aqueous carrier is about 8 mg/mL, 9 mg/mL, 10mg/mL, 12 mg/mL, 15 mg/mL or more.

In some embodiments, the insulin-Fc fusion protein is administered as abolus, infusion, or an intravenous push. In some embodiments, the fusionprotein is administered through syringe injection, pump, pen, needle, orindwelling catheter. In some embodiments, the insulin-Fc fusion proteinis administered by a subcutaneous bolus injection. Methods ofintroduction may also be provided by rechargeable or biodegradabledevices. Various slow release polymeric devices have been developed andtested in vivo in recent years for the controlled delivery of drugs,including proteinaceous biopharmaceuticals. A variety of biocompatiblepolymers (including hydrogels), including both biodegradable andnon-degradable polymers, can be used to form an implant for thesustained release of a compound at a particular target site.

Dosages

Actual dosage levels of the insulin-Fc fusion protein can be varied soas to obtain an amount of the active ingredient that is effective toachieve the desired therapeutic response for a particular subject (e.g.dog or cat). The selected dosage level will depend upon a variety offactors including the activity of the particular fusion proteinemployed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular fusion protein employed, the age, sex, weight, condition,general health and prior medical history of the subject being treated,and like factors well known in the medical arts.

In general, a suitable dose of an insulin-Fc fusion protein will be theamount that is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above. Generally, intravenous and subcutaneous doses of theinsulin-Fc fusion protein for a dog or cat will range from about 0.001to about 1 mg per kilogram (e.g. mg/kg) of body weight per day, e.g.,about 0.001 to 1 mg/kg/day, about 0.01 to 0.1 mg/kg/day, about 0.1 to 1mg/kg/day, or about 0.01 to 1 mg/kg/day. In still other embodiments, thefusion protein is administered at a dose between 0.025 and 4 mg perkilogram of body weight per week, e.g., between 0.025 and 0.5mg/kg/week.

The present disclosure contemplates formulation of the insulin-Fc fusionprotein in any of the aforementioned pharmaceutical compositions andpreparations. Furthermore, the present disclosure contemplatesadministration via any of the foregoing routes of administration. One ofskill in the art can select the appropriate formulation and route ofadministration based on the condition being treated and the overallhealth, age, and size of the patient being treated.

EXAMPLES

The present technology is further illustrated by the following Examples,which should not be construed as limiting in any way.

General Methods, Assays, and Materials Example 1: Synthesis and Methodsof Making an Insulin-Fc Fusion Protein in HEK293 Cells

Insulin-Fc fusion proteins were synthesized as follows. A gene sequenceof interest was constructed using proprietary software (LakePharma,Belmont, Calif.) and was cloned into a high expression mammalian vector.HEK293 cells were seeded in a shake flask 24 hours before transfectionand were grown using serum-free chemically defined media. A DNAexpression construct that encodes the insulin-Fc fusion protein ofinterest was transiently transfected into a suspension of HEK293 cellsusing the (LakePharma, Belmont, Calif.) standard operating procedure fortransient transfection. After 20 hours, the cells were counted todetermine the viability and viable cell count, and the titer wasmeasured by ForteBio® Octet® (Pall ForteBio LLC, Fremont, Calif.).Additional readings were taken throughout the transient transfectionproduction run. The culture was harvested on or after day 5.

Example 2: Synthesis and Methods of Making an Insulin-Fc Fusion Proteinin CHO Cells

A CHO cell line was originally derived from CHO-K1 (LakePharma, Belmont,Calif.), and the endogenous glutamine synthetase (GS) genes were knockedout by recombinant technology using methods known in the art. Stableexpression DNA vectors were designed and optimized for CHO expressionand GS selection and incorporated into a high expression mammalianvector (LakePharma, Belmont, Calif.). The sequence of each completedconstruct was confirmed prior to initiating scale up experiments. Thesuspension-adapted CHO cells were cultured in a humidified 5% CO₂incubator at 37° C. in a chemically defined media (CD OptiCHO;Invitrogen, Carlsbad, Calif.). No serum or other animal-derived productswere used in culturing the CHO cells.

Approximately 80 million suspension-adapted CHO cells, growing in CDOptiCHO media during the exponential growth phase, were transfected byelectroporation using MaxCyte® STX® system (MaxCyte, Inc., Gaithersburg,Md.) with 80 μg DNA to a create a stable CHO cell line for eachinsulin-Fc fusion protein (DNA construct contains the full-lengthsequence of the insulin-Fc fusion protein). After twenty-four hours, thetransfected cells were counted and placed under selection for stableintegration of the insulin-Fc fusion genes. The transfected cells wereseeded into CD OptiCHO selection media containing between 0-100 μMmethionine sulfoximine (MSX) at a cell density of 0.5×10⁶ cells/mL in ashaker flask and incubated at 37° C. with 5% CO₂. During a selectionprocess, the cells were spun down and resuspended in fresh selectionmedia every 2-3 days until the CHO stable pool recovered its growth rateand viability. The cell culture was monitored for growth and titer.

The cells were grown to 2.5×10⁶ cells per mL. At the time of harvest forcell banking, the viability was above 95%. The cells were thencentrifuged, and the cell pellet was resuspended in the CD OptiCHO mediawith 7.5% dimethyl sulfoxide (DMSO) to a cell count of 15×10⁶ cells permL per vial. Vials were cryopreserved for storage in liquid nitrogen.

A small-scale-up production was performed using the CHO cells asfollows. The cells were scaled up for production in CD OptiCHO growthmedium containing 100 μM MSX at 37° C. and fed every 2-4 days as needed,with CD OptiCHO growth medium supplemented with glucose and additionalamino acids as necessary for approximately 14-21 days. The conditionedmedia supernatant harvested from the stable pool production run wasclarified by centrifuge spinning The protein was run over a Protein A(MabSelect, GE Healthcare, Little Chalfont, United Kingdom) columnpre-equilibrated with binding buffer. Washing buffer was then passedthrough the column until the OD280 value (NanoDrop, Thermo Scientific)was measured to be at or near background levels. The insulin-Fc fusionprotein was eluted using a low pH buffer, elution fractions werecollected, and the OD280 value of each fraction was recorded. Fractionscontaining the target insulin-Fc fusion protein were pooled andoptionally further filtered using a 0.2 μM membrane filter.

The cell line was optionally further subcloned to monoclonality andoptionally further selected for high titer insulin-Fc-fusionprotein-expressing clones using the method of limiting dilution, amethod known to those skilled in the art. After obtaining a high titer,monoclonal insulin-Fc fusion protein-expressing cell line, production ofthe insulin-Fc fusion protein was accomplished as described above ingrowth medium without MSX, or optionally in growth medium containingMSX, to obtain a cell culture supernatant containing the recombinant,CHO-made, insulin-Fc fusion protein. The MSX concentration wasoptionally increased over time to exert additional selectivity forclones capable of yielding higher product titers.

Example 3: Purification of an Insulin-Fc Fusion Protein

Purification of an insulin-Fc fusion protein was performed as follows.Conditioned media supernatants containing the secreted insulin-Fc fusionprotein were harvested from the transiently or stably transfected HEKproduction runs and were clarified by centrifugation. The supernatantcontaining the desired insulin-Fc fusion protein was run over a ProteinA or a Protein G column and eluted using a low pH gradient. Optionally,recovery of the insulin-Fc fusion proteins could be enhanced byreloading of the initial Protein A or Protein G column eluent again ontoa second Protein A or Protein G column. Afterwards, the eluted fractionscontaining the desired protein were pooled and buffer exchanged into 200mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0 buffer. A final filtrationstep was performed using a 0.2 pin membrane filter. The final proteinconcentration was calculated from the solution optical density at 280nm. Further optional purification by ion-exchange chromatography (e.g.using an anion exchange bead resin or a cation exchange bead resin), gelfiltration chromatography, or other methods was performed as necessary.

Example 4: Structure Confirmation by Non-Reducing and Reducing CE-SDS

Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) analysis wasperformed in a LabChip® GXII (Perkin Elmer, Waltham, Mass.) on asolution of a purified insulin-Fc fusion protein dissolved in 200 mMHEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0 buffer, and the electropherogramwas plotted. Under non-reducing conditions, the sample was run againstknown molecular weight (MW) protein standards, and the eluting peakrepresented the ‘apparent’ MW of the insulin-Fc fusion proteinhomodimer.

Under reducing conditions (e.g. using beta-mercaptoethanol to breakdisulfide bonds of the insulin-Fc fusion homodimer), the apparent MW ofthe resulting insulin-Fc fusion protein monomer is compared against halfthe molecular weight of the insulin-Fc fusion protein homodimer as a wayof determining that the structural purity of the insulin-Fc fusionprotein is likely to be correct.

Example 5: Sequence Identification by LC-MS with Glycan Removal

To obtain an accurate estimate of the insulin-Fc mass via massspectroscopy (MS), the sample is first treated to remove naturallyoccurring glycan that might interfere with the MS analysis. 100 μL of a2.5 mg/mL insulin-Fc fusion protein dissolved in 200 mM HEPES, 100 mMNaCl, 50 mM NaOAc, pH 7.0 buffer solution is first buffer exchanged into0.1 M Tris, pH 8.0 buffer containing 5 mM EDTA using a Zeba desaltingcolumn (Pierce, ThermoFisher Scientific, Waltham, Mass.). 1.67 μL ofPNGase F enzyme (Prozyme N-glycanase) is added to this solution in orderto remove N-linked glycan present in the fusion protein (e.g., glycanlinked to the side chain of the asparagine located at the cNg-N site),and the mixture is incubated at 37° C. overnight in an incubator. Thesample is then analyzed via LC-MS (NovaBioassays, Woburn, Mass.)resulting in a molecular mass of the molecule which corresponds to thedesired homodimer without the glycan. This mass is then furthercorrected since the enzymatic process used to cleave the glycan from thecNg-asparagine also deaminates the asparagine side chain to form anaspartic acid, and in doing so the enzymatically treated homodimer gains2 Da overall, corresponding to a mass of 1 Da for each chain present inthe homodimer. Therefore, the actual molecular mass is the measured massminus 2 Da to correct for the enzymatic modification of the insulin-Fcfusion protein structure in the analytical sample.

Example 6: % Homodimer by Size-Exclusion Chromatography

Size-exclusion chromatography (SEC-HPLC) of insulin-Fc fusion proteinswas carried out using a Waters 2795HT HPLC (Waters Corporation, Milford,Mass.) connected to a 2998 Photodiode array at a wavelength of 280 nm.100 μL or less of a sample containing an insulin-Fc fusion protein ofinterest was injected into a MAbPac SEC-1, 5 μm, 4×300 mm column(ThermoFisher Scientific, Waltham, Mass.) operating at a flow rate of0.2 mL/min and with a mobile phase comprising 50 mM sodium phosphate,300 mM NaCl, and 0.05% w/v sodium azide, pH 6.2. The MAbPac SEC-1 columnoperates on the principle of molecular size separation. Therefore,larger soluble insulin-Fc aggregates (e.g. multimers of insulin-Fcfusion protein homodimers) eluted at earlier retention times, and thenon-aggregated homodimers eluted at later retention times. In separatingthe mixture of homodimers from aggregated multimeric homodimers viaanalytical SEC-HPLC, the purity of the insulin-Fc fusion proteinsolution in terms of the percentage of non-aggregated homodimer wasascertained.

Example 7: In Vitro IM-9 Insulin Receptor Binding of an ExemplaryInsulin-Fc Fusion Protein at 4° C.

Human IM-9 cells (ATTC #CCL-159) that express human insulin receptorwere cultured and maintained in complete RPMI 5% FBS medium at 70-80%confluency. Cultures of IM-9 cells were centrifuged at 250×g (˜1000 rpm)for 10 min to pellet the cells. Cells were washed once with HBSS or PBSbuffer, resuspended in cold FACS staining medium (HBSS/2 mM EDTA/0.1%Na-azide+4% horse serum) to a concentration of 8×10⁶ cells/mL and kepton ice or 4° C. until test solutions were made. The insulin-Fc proteinwas diluted in FACS buffer in 1:3 serial dilutions as 2× concentrationsin 1.2 mL tubes (approx. 60 μL volume of each dilution), and thesolutions were kept cold on ice until ready for pipetting.

Biotinylated-RHI was diluted in FACS staining medium to a concentrationof 1.25 μg/mL. 40 μL of the serially diluted test compound and 8 μL of1.25 μg/mL Biotin-RHI were added into each well of a V bottom microtiterplate, mixed by slow vortexing, and placed on ice. 40 μL of an IM-9 cellsuspension (8×10⁶ cells/mL) was then added to each well by multichannelpipette, mixed again gently and incubated on ice for 30 min to allowcompetitive binding on the insulin receptor on IM-9 cells. Cells werethen washed twice with 275 μL of ice-cold FACS wash buffer (HBSS/2 mMEDTA/0.1% Na-azide+0.5% horse serum) by centrifuging the V-bottom plateat 3000 rpm for 3 min and aspirating the supernatant. Cells were thenresuspended in 40 μL of FACS staining medium containing 1:100 dilutedStreptavidin-PE (Life Technologies) for 20 min on ice. Cells were thenwashed once with 275 μL of ice-cold FACS buffer and finally fixed with3% paraformaldehyde for 10 min at room temp. Cells were then washed oncewith 275 μL of ice-cold FACS buffer and resuspended in 250 μl of FACSbuffer for analysis.

The V-bottom plates containing cells were then analyzed on a Guava 8-HTflow cytometer (Millipore). Biotinylated-RHI binding to insulin receptorwas quantitated by the median fluorescence intensity (MFI) of the cellson the FACS FL-2 channel for each concentration of the test compound.Control wells were labeled only with biotinylated-RHI and were used tocalculate the percent (%) inhibition resulting from each test compoundconcentration. The % inhibition by test compounds of biotinylated-RHIbinding on IM-9 cells was plotted against log concentrations of the testcompound, and the resulting IC50 values were calculated using GraphPadPrism (GraphPad Software, La Jolla, Calif.) for the test compounds.Lower IC50 values of the test compound therefore indicate greater levelsof biotinylated-RHI inhibition at lower concentrations indicatingstronger binding of the insulin-Fc fusion protein to the insulinreceptor. A control compound, such as unlabeled recombinant humaninsulin (RHI) was also used as an internal standard to generate an RHIIC50 against which a given compound IC50 could be ratioed(IC50(compound)/IC50(RHI)). Lower IC50 ratios have more similar bindingto RHI (stronger binding to insulin receptor), while higher IC50 ratioshave weaker binding to the insulin receptor relative to RHI.

Example 8: In Vitro Fc(Gamma) Receptor I Binding Affinity Assay

The binding of insulin-Fc fusion proteins to the Fc(gamma) receptor I atpH 7.4 was conducted using an ELISA assay as follows. Since neithercanine nor feline Fc(gamma) receptor I was not commercially available,human Fc(gamma) receptor I (i.e., rhFc(gamma) receptor I) was used as asurrogate mammalian receptor. Insulin-Fc compounds were diluted to 10μg/mL in sodium bicarbonate buffer at pH 9.6 and coated on Maxisorp(Nunc) microtiter plates overnight at 4° C., after which the microplatestrips were washed 5 times with PBST (PBS/0.05% Tween-20) buffer andblocked with Superblock blocking reagent (ThermoFisher). Serialdilutions of biotinylated rhFc(gamma) receptor I (recombinant humanFc(gamma)R-I; R&D Systems) were prepared in PBST/10% Superblock bufferfrom 6000 ng/mL to 8.2 ng/mL and loaded at 100 μL/well onto themicroplate strips coated with insulin-Fc fusion protein. The microtiterplate was incubated for 1 hour at room temperature after which themicroplate strips were washed 5 times with PBST and then loaded with 100μL/well of streptavidin-HRP diluted 1:10000 in PBST/10% Superblockbuffer. After incubating for 45 min, the microplate strips were washedagain 5 times with PB ST. TMB was added to reveal the bound Fc(gamma)receptor I proteins and stopped with ELISA stop reagent (BostonBioproducts). The plate was read in an ELISA plate reader at 450 nm, andthe OD values (proportional to the binding of rhFc(gamma) receptor I toinsulin-Fc protein) were plotted against log concentrations ofrhFc(gamma) receptor I added to each well to generate binding curvesusing GraphPad Prism software.

Example 9: In Vitro Measurement of Insulin-Fc Fusion Protein Affinityfor the Canine FcRn Receptor

In vitro binding affinity of insulin-Fc fusion proteins containing Fcfragments of canine or feline IgG origin to the canine FcRn receptor wasmeasured via an ELISA technique conducted at a solution pH of 5.5. Theslightly acidic pH is the preferred binding environment for Fcfragment-containing molecules to bind to the FcRn receptor. In vivo,cells express FcRn on their surfaces and internally in the endosomes. Asmolecules containing Fc fragments are brought into the cell throughnatural processes (e.g. pinocytosis or endocytosis), the pH changes to alower pH in the endosomes, where the FcRn receptor binds to Fcfragment-containing molecules that would otherwise be degraded in theendosomal-lysosomal compartments, thereby allowing these molecules torecycle back to the cellular surface where the pH is closer to neutral(e.g., pH 7.0-7.4). Neutral pH disfavors binding to the FcRn receptorand allows release of the Fc-fragment containing molecules back intocirculation. This is a primary mechanism by which Fc fragment-containingmolecules exhibit prolonged circulatory pharmacokinetic half-lives invivo.

Insulin-Fc fusion proteins comprising Fc fragments of canine or felineorigin were diluted to 10 μg/ml in sodium bicarbonate pH 9.6 buffer andcoated in duplicate on Maxisorb ELISA plate strips for 1-2 hours at RT.The strips were then washed 4 times with PBST (PBS/0.1% Tween-20) bufferand blocked with Superblock blocking reagent (ThermoFisher). Strips forFcRn binding were then washed again twice with pH 5.5 MES/NaCl/Tween (50mM MES/150 mM NaCl/0.1% Tween-20) buffer before addition of the FcRnreagent (biotinylated canine FcRn; Immunitrack). Since no feline FcRnreagent was found to be commercially available, insulin-Fc fusionproteins containing either a canine Fc fragment or a feline Fc fragmentwere assayed for binding to the canine FcRn. Serial dilutions (1:3×dilutions) of biotinylated FcRn reagent were prepared in pH 5.5MES/NaCl/Tween/10% Superblock buffer at concentrations from 1000 ng/mlto 0.45 ng/ml and loaded at 100 μL/well using a multichannel pipettoronto the strips coated with the insulin-Fc fusion protein compounds. Theassay plate was then incubated for 1 hour at room temperature. FcRnbinding strips were washed 4 times with pH 5.5 MES/NaCl/Tween buffer andthen loaded with 100 μl/well streptavidin-HRP diluted 1:10000 in pH 5.5MES/NaCl/10% Superblock buffer. After incubating for 45 minutes, stripswere washed again 4 times with pH 5.5 MES/NaCl/Tween buffer. TMB wasfinally added to reveal the bound biotinylated-canine FcRn reagent, andthe color development was stopped with the ELISA stop reagent. The platewas read in an ELISA plate reader at a wavelength of 450 nm. The ODvalues (proportional to the binding of canine-FcRn to the insulin-Fcfusion protein test compounds) were plotted against log concentrationsof FcRn added to each well to generate binding curves using GraphPadPrism software. EC50 values for each binding curve were calculated tocompare between different compounds.

Example 10: Generalized Procedure for Determination of In VivoPharmacodynamics (PD) after Single Administration of Insulin Fc-FusionProteins in Dogs or Cats

Insulin-Fc fusion proteins were assessed for their effects on fastingblood glucose levels as follows. N=1, 2, 3 or more healthy,antibody-naïve, dogs weighing approximately 10-15 kg or cats weighingapproximately 5 kg were used, one for each insulin-Fc fusion protein.Animals were also observed twice daily for signs of anaphylaxis,lethargy, distress, pain, etc., and, optionally for some compounds,treatment was continued for an additional three weekly subcutaneousinjections or more to observe if the glucose lowering capability of thecompounds lessened over time, a key sign of potential induction ofneutralizing anti-drug antibodies. On day 0, the animals received asingle injection either via intravenous or subcutaneous administrationof a pharmaceutical composition containing an insulin Fc-fusion proteinhomodimer at a concentration between 1 and 10 mg/mL in a solution ofbetween 10-50 mM sodium hydrogen phosphate, 50-150 mM sodium chloride,0.005-0.05% v/v Tween-80, and optionally a bacteriostat (e.g. phenol,m-cresol, or methylparaben) at a concentration of between 0.02-1.00mg/mL, at a solution pH of between 7.0-8.0, at a dose of 0.08-0.80 mginsulin-Fc fusion protein/kg (or approximately equivalent to 1.2-12.3nmol/kg or approximately equivalent to 0.4-4.0 U/kg insulin equivalenton molar basis). On day 0, blood was collected from a suitable veinimmediately prior to injection and at 15, 30, 45, 60, 120, 240, 360, and480 min and at 1, 2, 3, 4, 5, 6, and 7 days post injection.

For each time point, a minimum of 1 mL of whole blood was collected. Aglucose level reading was immediately determined using a glucose meter(ACCU-CHEK® Aviva Plus), which required approximately one drop of blood.Average % fasting blood glucose levels (% FBGL) from day 0 to day 7 wereplotted to assess the bioactivity of a given insulin-Fc fusion protein.

Example 11: Generalized Procedure for Determination of In VivoPharmacodynamics (PD) after Repeated Administration of Insulin-Fc FusionProteins in Canines or Felines

Insulin-Fc fusion proteins were assessed for their effects on bloodglucose levels over repeated injections as follows. Healthy,antibody-naïve, dogs or cats weighing approximately between 5 and 20 kgwere used, and each animal was administered doses of an insulin-Fcfusion protein. Animals were observed twice daily for signs ofanaphylaxis, lethargy, distress, pain, and other negative side effects,and optionally for some compounds, treatment was continued for up to anadditional two to five subcutaneous injections to observe if the glucoselowering capability of the compounds decreased over time, indicating thepossible presence of neutralizing anti-drug antibodies in vivo. On day0, the animals received a single subcutaneous injection of apharmaceutical composition containing an insulin Fc-fusion protein in asolution of 10-50 mM sodium hydrogen phosphate, 50-150 mM sodiumchloride, 0.005-0.05% v/v Tween-80, and optionally a bacteriostat (e.g.phenol, m-cresol, or methylparaben) at a concentration of between0.02-1.00 mg/mL, at a solution pH of between 7.0-8.0, at a dose of0.08-0.80 mg insulin-Fc fusion protein/kg (or approximately equivalentto 1.2-12.3 nmol/kg or approximately equivalent to 0.4-4.0 U/kg insulinequivalent on molar basis). On day 0, blood was collected from asuitable vein immediately prior to injection and at 15, 30, 45, 60, 120,240, 360, and 480 min and at 1, 2, 3, 4, 5, 6, and 7 days postinjection.

Subsequent subcutaneous injections were given no more frequently thanonce-weekly, and in some cases the injections were given at differentintervals based on the pharmacodynamics of a given insulin-Fc fusionprotein formulation. Subsequent injections for each insulin-Fc fusionprotein were adjusted to higher or lower doses, depending on thedemonstrated pharmacodynamics of the insulin-Fc fusion protein. Forinstance, if the dose of a first injection on day 0 was found to beineffective at lowering blood glucose, the subsequent dose levels ofinjected insulin-Fc fusion protein were adjusted upward. In a similarmanner, if the dose of a first injection on day 0 was found to lowerglucose in too strong a manner, then subsequent dose levels of injectedinsulin-Fc fusion protein were adjusted downward. It was also found thatinterim doses or final doses could be adjusted in a similar manner asneeded. For each dose, blood was collected from a suitable vein justimmediately prior to injection and at 15, 30, 45, 60, 120, 240, 360, and480 min and at 1, 2, 3, 4, 5, 6, 7 days (and optionally 14 days) postinjection. For each time point, a minimum of 1 mL of whole blood wascollected. A glucose level reading was immediately determined using aglucose meter (ACCU-CHEK® Aviva Plus), which required approximately onedrop of blood. Average % fasting blood glucose levels (% FBGL) fromthroughout the study were plotted against time which allows thebioactivity of a fusion protein to be determined.

To determine the bioactivity of each dose, an area-over-the-curve (AOC)analysis was conducted as follows. After constructing the % FBGL versustime data, the data was then entered into data analysis software(GraphPad Prism, GraphPad Software, San Diego Calif.). The software wasused to first conduct an area-under-the curve analysis (AUC) tointegrate the area under the % FBGL vs. time curve for each dose. Toconvert the AUC data into the desired AOC data, the following equationwas used: AOC=TPA−AUC; where TPA is the total possible area obtained bymultiplying each dose lifetime (e.g., 7 days, 14 days, etc.) by 100%(where 100% represents the y=100% of the % FBGL vs. time curve). Forexample, given a dose lifetime of 7 days and a calculated AUC of 500%FBGL·days, gives the following for AOC: AOC=(100% FBGL×7 days)−(500%FBGL·days)=200% FBGL·days. The analysis can be performed for eachinjected dose in a series of injected doses to obtain the AOC values forinjection 1, injection 2, injection 3, etc.

As the doses of insulin-Fc fusion protein may vary as previouslydiscussed, it is often more convenient to normalize all calculated AOCvalues for a given insulin-Fc fusion protein to a particular dose ofthat insulin-Fc fusion protein. Doing so allows for convenientcomparison of the glucose-lowering potency of an insulin-Fc fusionprotein across multiple injections, even if the dose levels changeacross the injections of a given study. Normalized AOC (NAOC) for agiven dose is calculated as follows: NAOC=AOC/D with units of %FBGL·days·kg/mg; where D is the actual dose injected into the animal inmg/kg. NAOC values may be calculated for each injection in a series ofinjections for a given animal and may be averaged across a group ofanimals receiving the same insulin-Fc fusion protein formulation.

The NAOC ratio (NAOCR) may also be calculated for each injection in aseries of injections for a given animal by taking the NAOC values foreach injection (e.g. injections 1, 2, 3, . . . N) and dividing each NAOCfor a given injection by the NAOC from injection 1 as follows:NAOCR=(NAOC(Nth injection)/NAOC(injection 1)). By evaluating the NAOCRof a given insulin-Fc homodimer fusion protein formulation for the Nthinjection in a series of injections, it is possible to determine whetherthe in vivo glucose lowering activity of a given insulin-Fc fusionprotein has substantially retained its bioactivity over a series of Ndoses (e.g., NAOCR for the Nth dose of greater than 0.5) or whether thein vivo glucose lowering activity of a given insulin-Fc fusion proteinhas lost a substantial portion of its potency (e.g., NAOCR of the Nthdose is less than 0.5) over a course of N doses, indicating thepotential formation of neutralizing anti-drug antibodies in vivo. Inpreferred embodiments, the ratio of NAOC following the thirdsubcutaneous injection to the NAOC following the first subcutaneousinjection is greater than 0.5 (i.e., the NAOCR of the third subcutaneousinjection is greater than 0.5).

Example 12: Generalized Procedure for the Determination of In VivoPharmacokinetics (PK) in Canine and Feline Serum

An assay was constructed for measuring the concentrations of insulin-Fcfusion proteins comprising Fc fragments of a canine isotype in canineserum as follows. The assay comprises a sandwich ELISA format in whichtherapeutic compounds in serum samples are captured by ananti-insulin/proinsulin monoclonal antibody (mAb) coated on the ELISAplates and then detected by a HRP-conjugated anti-canine IgG Fc specificantibody followed by use of a TMB substrate system for colordevelopment. Maxisorp ELISA Plates (Nunc) are coated with theanti-insulin mAb clone D6C4 (Biorad) in coating buffer (pH=9.6 sodiumcarbonate-sodium biocarbonate buffer) at 5 μg/ml overnight at 4° C.Plates are then washed 5× with PBST (PBS+0.05% Tween 20) and blocked fora minimum of one hour at room temperature (or overnight at 4C) withSuperBlock blocking solution (ThermoFisher). Test serum samples arediluted to 1:20 in PBST/SB/20% HS sample dilution buffer (PBS+0.1% Tween20+10% SuperBlock+20% horse serum). For making a standard curve, theinsulin-Fc fusion protein of interest is diluted in sample dilutionbuffer (PBST/SB/20% HS)+5% of pooled beagle serum (BioIVT) from aconcentration range of 200 ng/ml to 0.82 ng/ml in 1:2.5 serialdilutions. Standards and diluted serum samples are added to the blockedplates at 100 μl/well in duplicate and are incubated for 1 hour at roomtemperature. Following incubation, samples and standards are washed 5×with PBST. HRP-conjugated goat anti-canine IgG Fc (Sigma) detectionantibody is diluted to about 1:15,000 in PBST/SB/20% HS buffer and 100μl is added to all the wells and incubated for 45 minutes at roomtemperature in the dark. Plates are washed 5× with PBST and once withdeionized water and developed by the addition of 100 μl/well TMB(Invitrogen) for 8-10 minutes at room temperature. Color development isthen stopped by the addition of 100 μl/well ELISA Stop Solution (BostonBioproducts) and the absorbance is read at 450 nm using a SpectraMaxplate reader (Molecular Devices) within 30 minutes. Concentrations ofinsulin-Fc fusion protein compounds in the samples are calculated byinterpolation on a 4-PL curve using SoftMaxPro software.

Similarly, an assay was constructed for measuring the concentrations ofinsulin-Fc fusion proteins comprising Fc fragments of a feline isotypein feline serum as follows. The assay comprises a sandwich ELISA formatin which therapeutic compounds in serum samples are captured by ananti-insulin/proinsulin mAb coated on the ELISA plates and then detectedby a HRP-conjugated goat anti-feline IgG Fc specific antibody followedby use of a TMB substrate system for color development. Maxisorp ELISAPlates (Nunc) are coated with the anti-insulin mAb clone D6C4 (Biorad)in coating buffer (pH=9.6 sodium carbonate-sodium biocarbonate buffer)at 5 μg/ml overnight at 4° C. Plates are then washed 5× with PBST(PBS+0.05% Tween 20) and blocked for a minimum of one hour at roomtemperature (or overnight at 4C) with SuperBlock blocking solution(ThermoFisher). Test serum samples are diluted to 1:20 in PBST/SB/20% HSsample dilution buffer (PBS+0.1% Tween 20+10% SuperBlock+20% horseserum). For making a standard curve, the insulin-Fc fusion proteincompound of interest is diluted in sample dilution buffer (PBST/SB/20%HS)+5% of normal cat serum (Jackson Immunoresearch) from a concentrationrange of 200 ng/ml to 0.82 ng/ml in 1:2.5 serial dilutions. Standardsand diluted serum samples are added to the blocked plates at 100 μl/wellin duplicate and are incubated for 1 hour at room temperature. Followingincubation, samples and standards are washed 5× with PBST.HRP-conjugated goat anti-feline IgG Fc (Bethyl Lab) detection antibodyis diluted to about 1:20,000 in PBST/SB/20% HS buffer and 100 μl isadded to all the wells and incubated for 45 minutes at room temperaturein the dark. Plates are washed 5× with PBST and once with deionizedwater and developed by the addition of 100 μl/well TMB (Invitrogen) for8-10 minutes at room temperature. Color development is then stopped bythe addition of 100 μl/well ELISA Stop Solution (Boston Bioproducts) andabsorbance is read at 450 nm using a SpectraMax plate reader (MolecularDevices) within 30 minutes. Concentrations of insulin-Fc fusion proteincompounds in the samples are calculated by interpolation on a 4-PL curveusing SoftMaxPro software.

Example 13: Assay Protocol for Measuring Anti-Drug Antibodies in CanineSerum

Maxisorp ELISA Plates (Nunc) are coated with the insulin-Fc fusionprotein of interest diluted in coating buffer (pH=9.6Carbonate-Biocarbonate buffer) at 10 μg/mL overnight at 4° C. formeasuring ADAs against the test compound. For measuring ADAs against theinsulin portion of the insulin-Fc fusion protein containing an Fcfragment of canine IgG origin, plates are coated with purified insulinat 30 μg/mL in coating buffer. Plates are then washed 5× with PBST(PBS+0.05% Tween 20) and blocked for at least 1 hour (or overnight) withSuperBlock blocking solution (ThermoFisher, Waltham Mass.). Forcalculating the ADAs in canine IgG units, strips are directly coatedwith 1:2 serial dilutions of canine IgG (Jackson ImmunoresearchLaboratories, West Grove Pa.) in pH=9.6 Carb-Biocarb coating buffer atconcentrations between 300-4.69 ng/ml overnight at 4° C. and used tocreate a 7-point pseudo-standard curve. The standards strip plates arealso washed and blocked with SuperBlock blocking solution for at least 1hour (or overnight).

Test serum samples are diluted to greater than or equal to 1:100(typically tested as 1:200) in PBST/SB/20% HS sample dilution buffer(PBS+0.1% Tween 20+10% SuperBlock+20% horse serum) and added to theinsulin-Fc fusion protein coated (or RHI coated) strips at 100 μL/wellin duplicate. Duplicate strips of canine IgG coated standard strips arealso added to each plate and filled with PBST/SB (PBS+0.1% Tween 20+10%SuperBlock) buffer at 100 μL/well. Plates are incubated for 1 hour at RTand then washed 5× with PB ST. For detection of ADAs, HRP-conjugatedGoat anti-feline IgG F(ab′)2 (anti-feline IgG F(ab′)2 reagent iscross-reacts to canine antibodies; Jackson Immunoresearch Laboratories,West Grove Pa.), which is diluted in PBST/SB to 1:10000 and added at 100μL/well to both sample and standard wells and incubated for 45 minutesat RT in dark. Plates are washed 5× with PBST and then one time withdeionized water and then developed by adding 100 μL/well TMB substrate(Invitrogen, ThermoFisher Scientific, Waltham Mass.) for 15-20 minutesat room temperature in the dark. Color development is then stopped byaddition of 100 μL/well of ELISA Stop Solution (Boston Bioproducts) andthe absorbance is read at 450 nm using a SpectraMax plate reader within30 minutes. The anti-drug antibody concentration is determined byinterpolating the OD values in the 4-PL pseudo-standard curve usingSoftMax Pro Software (Molecular Devices, San Jose Calif.).

To demonstrate the specificity of the detected ADAs, an “inhibition”assay is carried out. In the drug inhibition ADA assay, serum samplesare diluted 1:100 in PBST/SB/20% HS buffer and mixed with an equalvolume of 300 μg/mL of the relevant therapeutic compound (final sampledilution at 1:200 and final inhibitory compound at 150 μg/mL) andincubated for 30-40 minutes at room temperature to allow anti-drugantibodies to bind the free inhibitor (i.e., the therapeutic compound).After pre-incubation, the samples are added to insulin-Fc fusion proteincoated (or RHI coated) strips at 100 μL/well in duplicate. Samplesdiluted 1:200 in PBST/SB/20% HS buffer without the inhibitory compoundare also tested in the sample plates along with duplicate strips ofcanine IgG coated standards. Remaining steps of the assay procedure arecarried out as described above. The ADAs measured in the drug-inhibitedwells are matched with the non-inhibited ADA concentrations to assessthe specificity of the ADAs. If significant inhibition of ADA signals isobserved in the drug-inhibited wells, this means the ADAs are specificto the therapeutic compound.

Example 14: Assay Protocol for Measuring Anti-Drug Antibodies in FelineSerum

Maxisorp ELISA Plates (Nunc) are coated with the insulin-Fc fusionprotein of interest diluted in coating buffer (pH=9.6Carbonate-Biocarbonate buffer) at 10 μg/mL overnight at 4° C. formeasuring ADAs against the insulin-Fc fusion protein containing an Fcfragment of feline IgG origin. For measuring ADAs against the insulinportion of the insulin-Fc fusion protein, plates are coated withpurified insulin at 30 μg/mL in coating buffer. Plates are then washed5× with PBST (PBS+0.05% Tween 20) and blocked for at least 1 hour (orovernight) with SuperBlock blocking solution (ThermoFisher, WalthamMass.). For calculating the ADAs in feline IgG units, strips aredirectly coated with 1:2 serial dilutions of feline IgG (JacksonImmunoresearch Laboratories, West Grove Pa.) in pH=9.6 sodiumcarbonate-sodium bicarbonate coating buffer at concentrations between300-4.69 ng/mL overnight at 4° C. and used to create a 7-pointpseudo-standard curve. The standards strip plates are also washed andblocked with SuperBlock blocking solution for at least 1 hour (orovernight).

Test serum samples are diluted to greater than or equal to 1:100(typically tested as 1:200) in PBST/SB/20% HS sample dilution buffer(PBS+0.1% Tween 20+10% SuperBlock+20% horse serum) and added to theinsulin-Fc fusion protein coated (or RHI coated) strips at 100 μL/wellin duplicate. Duplicate strips of feline IgG coated standard strips arealso added to each plate and filled with PBST/SB (PBS+0.1% Tween 20+10%SuperBlock) buffer at 100 μL/well. Plates are incubated for 1 hour atroom temperature and then washed 5× with PBST. For detection of ADAs,HRP-conjugated goat anti-feline IgG F(ab′)2 (Jackson ImmunoresearchLaboratories, West Grove Pa.) is diluted in PBST/SB by a factor of1:10000 and added at 100 μL/well to both sample and standard wells andincubated for 45 minutes at room temperature in the dark. Plates arewashed 5× with PBST and one time with deionized water and developed bythe adding 100 μL/well TMB substrate (Invitrogen) for 15-20 minutes atroom temperature in the dark. Color development is then stopped byaddition of 100 μL/well of ELISA Stop Solution (Boston Bioproducts,Ashland Mass.) and the absorbance is read at 450 nm using a SpectraMaxplate reader within 30 minutes. Anti-drug antibody concentration isdetermined by interpolating the OD values in the 4-PL pseudo-standardcurve using SoftMax Pro Software (Molecular Devices, San Jose Calif.).

Example 15: Assay Procedure for Immunogenic Epitope Identification

Maxisorp ELISA microplates (Nunc) are coated with a library ofinsulin-Fc fusion protein homodimer compounds with known amino acidsequences, and the coated plates are blocked in a similar manner asdescribed in the anti-drug antibody ELISA assay Examples 13 and 14,except that each compound in the library is coated on a separateindividual strip of ELISA microplate wells. The compounds in the librarycomprise a range of insulin-Fc fusion proteins with different insulinpolypeptide amino acid compositions, including various B-chain, C-chain,and A-chain amino acid mutations, different linker compositions, anddifferent Fc fragment compositions, including some of human origin.Separately, some plate strip wells are directly coated with 1:2 serialdilutions of canine or feline IgG (Jackson Immunoresearch Laboratories,West Grove Pa.) for calculating the anti-drug antibodies (ADA) in canineor feline IgG units, respectively, as described in Examples 13 and 14.

Serum obtained from individual dogs or cats receiving repeated doses ofan insulin-Fc fusion protein is first screened on the anti-drug antibodyELISA assay (Example 13 for dogs and Example 14 for cats). Serum samplesdemonstrating moderate or high positivity (e.g. moderate or high titersof antibodies) on the assay of Example 13 or Example 14 are seriallydiluted (1:200 to 1:8000) in PBST/SB/20% HS sample dilution buffer(PBS+0.1% Tween 20+10% SuperBlock+20% horse serum) and added to theplates coated with the library of insulin-Fc fusion protein compoundsfor 1 hour at RT. Following incubation, the plates are washed 5 timeswith PBST. For detection of canine or feline antibodies capable ofcross-reacting to the coated compound library, HRP conjugated goatanti-feline IgG F(ab′)2 (Jackson Immunoresearch Laboratories, West GrovePa.), which is cross-reactive to both canine and feline IgGs, is dilutedin PBST/SB to 1:10000 and added at 100 μL/well to both sample andstandard wells and incubated for 45 min at RT in the dark. Plates arewashed 5 times with PBST, once with deionized water, and developed bythe adding 100 μL/well TMB substrate (Invitrogen, ThermoFisherScientific, Waltham Mass.) for 15-20 min at RT in the dark. Colordevelopment is then stopped by addition of 100 μL/well of ELISA StopSolution (Boston Bioproducts, Ashland Mass.) and absorbance is read at450 nm using a SpectraMax plate reader within 30 min. Anti-compoundcross-reactive antibody concentrations present in the serum samples aredetermined by interpolating the OD values in the 4-PL pseudo-standardcurve against the directly coated canine or feline IgG antibody controlsusing SoftMax Pro Software (Molecular Devices, San Jose Calif.).

By correlating the resulting antibody concentrations from the assay withthe known amino acid compositions of the coated insulin-Fc fusionprotein library, one can determine whether particular amino acidmutations or epitopes are responsible for causing none, some, most, orall of the total antibody signal on the assay, indicating no binding,weak binding, or strong binding to various insulin-Fc fusion proteinhomodimers. The mutations or epitopes responsible for moderate or strongbinding are herein referred to as immunogenic “hot spots”.

Example 16: Design Process for Obtaining Insulin-Fc Fusion Proteins withHigh Homodimer Titers and Acceptable Levels of Acute and Repeated DoseBioactivity in the Target Species

The process for meeting the design goals described in the DetailedDescription of the Invention comprised the following steps. First, theinsulin polypeptide of SEQ ID NO: 4 or SEQ ID NO: 5 was combined with aspecies-specific Fc fragment of a particular IgG isotype and a linkersuch that the resulting insulin-Fc fusion protein was most likely toyield a long acting bioactivity product with minimal immunogenicity(e.g., a species-specific IgG isotype was chosen with minimalFc(gamma)receptor I binding). The DNA sequence coding for the desiredfusion protein was prepared, cloned into a vector (LakePharma, SanCarlos, Calif.), and the vector was then used to transiently transfectHEK cells according to the procedure described in Example 1. Theinsulin-Fc fusion protein was then purified according to Examples 3 andthe overall protein yield and % homodimer measured according to Example6. Only candidates with a homodimer titer of greater than 50 mg/L wereconsidered acceptable, because titers less than this level are notlikely to result in commercial production titers that meet thestringently low manufacturing cost requirements for veterinary products.Selected insulin-Fc fusion proteins were then screened for indicators ofbioactivity through in vitro insulin receptor binding studies asdescribed in Example 7. Based on experience, only compounds thatexhibited IR activity IC50 values less than 5000 nM were deemed likelyto exhibit bioactivity in the target species. Although the in vitro IRIC50 value is a useful qualitative screening tool, it utilizes humanIM-9 cells which express the human insulin receptor and therefore it maynot capture some of the small differences in affinity between the canineor feline IR and the human IR. Furthermore, factors other than insulinreceptor binding may influence a compound's bioactivity in vivo (e.g.,affinity for canine or feline FcRn to allow for extended pharmacokineticelimination half-lives in vivo). Therefore, selected insulin-Fc fusionproteins that were acceptable from a manufacturing and IR activity IC50value standpoint were further screened for bioactivity in the animal ofinterest (e.g., dog or cat) to screen out any materials with less thanthe desired potency and/or duration of bioactivity (e.g., NAOC of lessthan 150% FBGL·days·kg/mg). Again, based on experience, at NAOC valuesof greater than 150% FBGL·days·kg/mg, the dose requirements in thetarget species will be sufficiently low so as to reach an acceptabletreatment cost. Lastly, an additional evaluation criterion was addedwhich is mentioned rarely if ever in the art. As discussed in moredetail in the Examples below, many insulin-Fc fusion protein embodimentsthat exhibit acceptable NAOC levels in the target species after thefirst dose, unexpectedly fail to maintain that level of bioactivityafter repeated doses. Furthermore, in most cases the reduction inrepeated dose bioactivity in the target species is correlated with thedevelopment of neutralizing anti-drug antibodies. This propensity togenerate anti-drug antibodies and the failure to maintain activityrender such insulin-Fc fusion proteins impractical for use in treating achronic disease such as canine diabetes or feline diabetes. Therefore,only the insulin-Fc fusions proteins exhibiting acceptable levels ofrepeated dose bioactivity (e.g., NAOCR values greater than 0.50 for thethird dose relative to the first dose) with minimal levels of anti-drugantibodies were deemed acceptable for use in the present invention.

Results—Insulin-Fc Fusion Proteins Comprising a Canine Fc FragmentExample 17: Canine Insulin-Fc Fusion Protein Comprising the Canine FcIgGA Isotype

An attempt was made to produce an insulin-Fc fusion protein comprisingthe insulin polypeptide sequence of SEQ ID NO: 5 and the Fc fragment ofthe canine IgGA isotype (SEQ ID NO: 15) using the peptide linker of SEQID NO: 12. The full amino acid sequence for the resulting insulin-Fcfusion protein is as follows:

(SEQ ID NO: 42) FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSPG

The insulin-Fc fusion protein of SEQ ID NO: 42 was synthesized in HEKcells according to Example 1 and purified according to Example 3. Theprotein yield was 22 mg/L after the Protein A purification step. Thestructure of the insulin-Fc fusion protein was confirmed according toExample 4 by non-reducing and reducing CE-SDS, and the sequence wasfurther identified by LC-MS with glycan removal according to Example 5.The % homodimer was measured by size-exclusion chromatography accordingto Example 6 and determined to be 24%, indicating a high degree ofhomodimer aggregates. The resulting homodimer titer was therefore only 5mg/L. In summary, manufacturing of the insulin-Fc fusion protein of SEQID NO: 42 in HEK cells resulted in a high level of aggregates and a lowhomodimer titer (5 mg/L), which did not meet the design goal of ahomodimer titer of greater than 50 mg/L.

Nevertheless, the insulin-Fc fusion protein of SEQ ID NO: 42 asevaluated for bioactivity. First, the insulin receptor binding of theinsulin-Fc fusion protein of SEQ ID NO: 42 was measured according toExample 7, resulting in an IC50 value of 2,733 nM indicating that thecompound is likely to be bioactive in vivo (i.e. IC50 less than 5000nM).

Next, the in vivo pharmacodynamics (PD) of the insulin-Fc fusion proteinof SEQ ID NO: 42 was measured after a single intravenous administrationof the compound to N=3 canines, according to Example 10. FIG. 2 showsthe percent fasting blood glucose level of SEQ NO: 42 as a function oftime. The NAOC for SEQ ID NO: 42 was calculated to be 105%FBGL·days·kg/mg according to the procedure of Example 11. The in vivohalf-life of SEQ ID NO: 42 was calculated to be less than one day usingthe method of Example 12. The relatively low NAOC was likely the resultof the high amount of aggregates in the sample (i.e., low % homodimer),but what soluble homodimer remained in circulation still only had apharmacokinetic elimination half-life of less than one day which wasdeemed unlikely support of once-weekly administration.

Example 18: Mutations of the Fc Fragment Region of Insulin-Fc FusionProteins Comprising the Canine IgGA Isotype

In an attempt to increase the % homodimer content, improve thebioactivity, and increase the half-life of the insulin-Fc fusion proteinof SEQ ID NO: 42, mutations were inserted into the Fc fragment CH3region to try to prevent intermolecular association (e.g., Fcfragment-Fc fragment interactions between molecules) and encouragestronger binding to the FcRn receptor (e.g., higher affinity for theFcRn) to increase recycling and systemic circulation time. The followinginsulin-Fc fusion proteins were synthesized in HEK cells according toExample 1, purified according to Examples 3, and tested according toExamples 4-7, which are shown below. The sequence alignment of SEQ IDNOs: 44, 46, 48, and 50 against SEQ ID NO: 42 and the differences inamino acid sequences are shown in FIG. 3.

(SEQ ID NO: 44) FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVLHEALHSHYTQKSLSLSPG (SEQ ID NO: 46)FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVLHETLQSHYTDLSLSHSPG (SEQ ID NO: 48)FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQSHYTDLSLSHSPG (SEQ ID NO: 50)FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVLHETLQNHYTDLSLSHSPG

The insulin-Fc fusion proteins based on canine IgGA variants are listedin Table 2 along with the corresponding protein yields, % homodimer, andhomodimer titers. The results show that the various mutations to theIgGA Fc fragment, instead of improving the % homodimer and homodimertiter, gave rise to highly aggregated proteins with extremely lowhomodimer titers of less than 5 mg/L. As such, the in vivo bioactivityand pharmacokinetics of the compounds could not be evaluated.

TABLE 2 Homodimer titers for sequences utilizing a native or mutatedcanine IgGA Fc fragment CH3 region Protein Yield Homodimer Titer SEQ IDNO: (mg/L) % Homodimer (mg/L) SEQ ID NO: 42 22 24%  5 SEQ ID NO: 44 330% 0 SEQ ID NO: 46 57 0% 0 SEQ ID NO: 48 67 0% 0 SEQ ID NO: 50 80 0% 0

Example 19: Canine Insulin-Fc Fusion Protein Using Other Canine FcFragment Isotypes

As described above, canine IgGA is thought to be the preferred isotypefor the Fc fragment to produce non-immunogenic insulin-Fc fusion proteinfor dogs due to its lack of Fc(gamma) I effector function in canines(much like the human IgG2 isotype in humans). However, insulin-Fc fusionproteins manufactured with a canine IgGA Fc fragment were highlyaggregated with an unacceptably low homodimer titer and unacceptably lowlevels of bioactivity and duration of action. Therefore, Fc fragmentsfrom the other canine IgG isotypes (canine IgGB of SEQ ID NO: 16),canine IgGC of SEQ ID NO: 17, and canine IgGD of SEQ ID NO: 18) wereevaluated as replacements for the canine IgGA Fc fragment of theinsulin-Fc fusion of SEQ ID NO: 42. The three insulin-Fc fusion proteinscontaining Fc fragments based on the canine IgGB, IgGC, and IgGDisotypes were synthesized using the same insulin polypeptide of SEQ IDNO: 5 and peptide linker of SEQ ID NO: 12 as were used to make theinsulin-Fc fusion protein of SEQ ID NO: 42. The proteins weremanufactured in HEK293 cells according to Example 1. The insulin-Fcfusion proteins were then purified using a Protein A column according toExample 3. The structures of the insulin-Fc fusion proteins wereconfirmed according to Example 4 by non-reducing and reducing CE-SDS,and the sequences were further identified by LC-MS with glycan removalaccording to Example 5. The % homodimer was measured by size-exclusionchromatography according to Example 6. Their sequences are shown belowand their sequence alignment comparison against SEQ ID NO: 42 is shownin FIG. 4:

(SEQ ID NO: 52) FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 54)FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGCNNCPCPGCGLLGGPSVFIFPPKPKDILVTARTPTVTCVVVDLDPENPEVQISWFVDSKQVQTANTQPREEQSNGTYRVVSVLPIGHQDWLSGKQFKCKVNNKALPSPIEEIISKTPGQAHQPNVYVLPPSRDEMSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEPESKYRMTPPQLDEDGSYFLYSKL SVDKSRWQRGDTFICAVMHEALHNHYTQISLSHSPG (SEQ ID NO: 56)FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGCISPCPVPESLGGPSVFIFPPKPKDILRITRTPEITCVVLDLGREDPEVQISWFVDGKEVHTAKTQPREQQFNSTYRVVSVLPIEHQDWLTGKEFKCRVNHIGLPSPIERTISKARGQAHQPSVYVLPPSPKELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPESKYHTTAPQLDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEALQNHYTDLSLSHSPGThe resulting protein yields, % homodimer, and homodimer titers aregiven in Table 3. Unexpectedly, only the insulin-Fc fusion protein ofSEQ ID NO: 52 comprising an Fc fragment based on the canine IgGB isotypedemonstrated a homodimer titer which met the design criteria of greaterthan 50 mg/L. The insulin-Fc fusion protein of SEQ ID NO: 54 comprisingan Fc fragment based on the canine IgGC isotype did not yield anycompound at all, and the insulin-Fc fusion protein of SEQ ID NO: 56comprising an Fc fragment based on the canine IgGD isotype demonstratedan appreciable protein yield but with a high degree of aggregation andtherefore an unacceptably low homodimer titer.

In vitro insulin receptor binding for the insulin-Fc fusion proteins ofSEQ ID NO: 52 and SEQ ID NO: 56 was tested according to the procedure ofExample 7. The insulin-Fc fusion protein of SEQ ID NO: 56 demonstratedan IC50 of greater than 5000 nM, indicating that the compound was highlyunlikely to show bioactivity in vivo. However, the insulin-Fc fusionprotein of SEQ ID NO: 52 demonstrated an IC50 of 28 nM indicating thatthis sequence was likely to be bioactive in vivo.

TABLE 3 Homodimer titers for sequences utilizing native canine IgGB,IgGC, and IgGD Fc fragments Protein Homodimer IR Binding, IgG YieldTiter IC50 SEQ ID NO: Fragment (mg/L) % Homodimer (mg/L) (nM) SEQ ID NO:42 IgGA 21 24% 5 2,733 (Example 17) SEQ ID NO: 52 IgGB 80 93% 74 28 SEQID NO: 54 IgGC 0  0% 0 DNM* SEQ ID NO: 56 IgGD 134 12% 16 >5000 *DNM =Did Not Measure

Example 20: In Vivo Efficacy of an Insulin-Fc Fusion Protein Comprisingthe Insulin Polypeptide of SEQ ID NO: 5 with a Canine IgGB Isotype FcFragment

Given the promising homodimer titer and insulin receptor activityresults in Example 19, the insulin-Fc fusion protein of SEQ ID NO: 52was tested for in vivo bioactivity according to Example 10 following anintravenous injection in each of N=3 healthy, antibody-naïve, beagledogs weighing approximately 10 kg. In a separate experiment, thecompound was administered subcutaneously to N=3 naïve beagle dogs. FIG.5 shows the % FBGL versus time for a single intravenous administrationof the insulin-Fc fusion protein of SEQ ID NO: 52, and FIG. 6 shows the% FBGL vs. time for a single subcutaneous administration of theinsulin-Fc fusion protein of SEQ ID NO: 52, both of which demonstratethat the insulin-Fc fusion protein of SEQ ID NO: 52 is significantlybioactive in dogs.

The NAOC was calculated according to the procedure of Example 11 todetermine the relative bioactivity and duration of action of theinsulin-Fc fusion protein. The NAOC of the insulin-Fc fusion protein ofSEQ ID NO: 52 injected intravenously was 399% FBGL·days·kg/mg which was3.8 times the NAOC of the insulin-Fc fusion protein of SEQ ID NO: 42injected intravenously, illustrating significantly increased bioactivityfor the insulin-Fc fusion protein comprising the canine IgGB Fc fragmentversus the insulin-Fc fusion protein comprising the canine IgGA Fcfragment. The NAOC of the insulin-Fc fusion protein of SEQ ID NO: 52injected subcutaneously was 366% FBGL·days·kg/mg, demonstrating a levelof bioactivity via subcutaneous administration that is similar to thatobtained via intravenous administration.

Example 21: In Vivo Immunogenicity Screening after Repeated SubcutaneousDoses of the Insulin-Fc Fusion Protein Comprising the InsulinPolypeptide of SEQ ID NO: 5 with a Canine IgGB Isotype Fc Fragment

Next, the repeated dose subcutaneous bioactivity of the insulin-Fcfusion protein of SEQ ID NO: 52 was tested in dogs as per the methoddescribed in Example 11. N=3 animals were dosed subcutaneously at day 0,at day 35, and at day 42, and the % FBGL was measured for the 7-daywindow after each dose according to Example 11. The NAOC and NAOCR werecalculated according to the procedure of Example 11 for each repeatedsubcutaneous injection. As illustrated in Table 4, repeated subcutaneousdosing in dogs unexpectedly revealed a significant decay in bioactivityby the third dose as measured by a significant decrease in the NAOCR(i.e., the NAOC for the third injection was only 0.40, or 40%, of theNAOC for the first injection).

TABLE 4 NAOC per dose and NAOCR for repeated doses of SEQ ID NO: 52Injection Number of NAOC NAOCR SEQ ID NO: 52 (% FBGL · days · kg/mg)(ratioed to Week 1) 1 330 1.0 2 339 1.1 3 115 0.4

Without being bound to any particular explanation, it was postulatedthat the cause of the significant reduction in bioactivity of theinsulin-Fc fusion protein of SEQ ID NO: 52 after the third repeatedsubcutaneous dose in dogs was due to the development of anti-drugantibodies that neutralized its biological activity. Anti-drugantibodies may be directed against the insulin polypeptide, linker, orFc-fragment portions of an insulin-Fc fusion protein. The immunogenicresponse manifests as interactions between antigen presenting cells,T-helper cells, B-cells, and their associated cytokines, which may leadto the production of endogenous antibodies against the drug (e.g.anti-drug antibodies). Binding antibodies are all isotypes capable ofbinding the insulin-Fc fusion protein, and these may be detected in animmunoassay as described in Example 13. Neutralizing antibodies thatinhibit functional activity of the insulin-Fc fusion protein aregenerally directed against an epitope that is required for bioactivity.To assess whether this was the case, serum that was collected prior tothe administration of each dose and at the end of the experimentdescribed in Examples 11 and 12 was tested to quantify the levels ofanti-drug antibodies according to Example 13. As shown in FIG. 7, levelsof anti-drug antibodies did indeed increase with multiple subcutaneousadministrations of the compound, indicating that the generation ofneutralizing anti-drug antibodies were the likely cause for thereduction in the NAOCR after the third injection of the insulinFc-fusion protein of SEQ ID NO: 52.

Example 22: Non-Glycosylated Insulin-Fc Fusion Protein Comprising theInsulin Polypeptide of SEQ ID NO: 5 with Canine IgGB Isotype FcFragments to Reduce the Potential Risk of Immunogenicity

As shown in Examples 19 and 20, the insulin-Fc fusion protein of SEQ IDNO: 52 showed acceptable % homodimer content, homodimer titer, andbioactivity in dogs; however, its use for a chronic disease such asdiabetes is compromised by the reduction in bioactivity (Example 21) andgeneration of anti-drug antibodies (Example 21) with repeatedsubcutaneous dosing. Without being bound to any particular theory, onepossible cause of the generation of anti-drug antibodies and thereduction in bioactivity is the increased interaction of the canine IgGBFc fragment with various receptors of the canine immune system (e.g.Fc(gamma) receptors, e.g. Fc(gamma)RI). Nevertheless, the canine IgGBisotype was the only one of the four canine IgG isotypes that, when usedfor the Fc fragment, resulted in an insulin-Fc fusion protein meetingthe manufacturability and single-dose bioactivity design goals (Example16). As described in the Detailed Description of the Invention, onemethod for reducing the Fc(gamma) interaction involves mutating the Fcfragment cNg site to prevent glycosylation during synthesis in the hostcell. Therefore, cNg site mutations were made to the Fc fragment regionof SEQ ID NO: 52 to reduce the binding affinity of the Fc fragment forFc(gamma) receptors in vivo, as measured by binding in an in vitro humanFc(gamma)RI assay described in Example 8. Verification of the lack ofglycan were performed using the LC-MS method of Example 5, but withomission of the PNGase F treatment step. The position of the cNg site inthe insulin-Fc fusion protein of SEQ ID NO: 52 is cNg-NB139. Mutationsto SEQ ID NO: 52 included SEQ ID NO: 58 comprising a mutation ofcNg-NB139-Q, SEQ ID NO: 60 comprising a mutation of cNg-NB139-S, SEQ IDNO: 62 comprising a mutation of cNg-NB139-D, and SEQ ID NO: 64comprising a mutation of cNg-NB139-K. The full amino acid sequences ofthe cNg-mutated insulin-Fc fusion proteins are listed below (with theNB139 position underlined) and the resulting sequence alignments areshown in FIG. 8 (Clustal Omega):

(SEQ ID NO: 58) FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFQGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 60)FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 62)FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFDGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 64)FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFKGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGThe insulin-Fc fusion proteins were manufactured in HEK293 cellsaccording to Example 1 and purified using a Protein A column accordingto Example 3. The structures of the insulin-Fc fusion proteins wereconfirmed according to Example 4 by non-reducing and reducing CE-SDS,and the sequences were further identified by LC-MS with glycan removalaccording to Example 5. The % homodimer was measured by size-exclusionchromatography according to Example 6. As shown in Table 5, thehomodimer titers of the insulin-Fc fusion proteins of SEQ ID NO: 60, SEQID NO: 62, and SEQ ID NO: 64 meet the design goal, while unexpectedlythe insulin-Fc fusion protein of SEQ ID NO: 58 containing thecNg-NB139-Q mutation did not meet the design goal for homodimer titer.

TABLE 5 Homodimer titers for cNg variations of SEQ ID NO: 52 ProteinHomodimer cNg Yield Titer SEQ ID NO: Mutation (mg/L) % Homodimer (mg/L)SEQ ID NO: 58 cNg-Q 37 98% 36 SEQ ID NO: 60 cNg-S 77 98% 75 SEQ ID NO:62 cNg-D 88 98% 86 SEQ ID NO: 64 cNg-K 68 98% 67

To determine which of the remaining three compounds was most likely toexhibit reduced immunogenicity, the Fc(gamma) receptor binding wasmeasured according to the procedure of Example 8. Low Fc(gamma) receptorbinding is most likely to correlate with minimum immunogenicity. Table 6compares the Fc(gamma) receptor I binding of these insulin-Fc fusionproteins with the Fc(gamma) receptor binding of the insulin-Fc fusionprotein of SEQ ID NO: 52 demonstrating unexpectedly that the insulin-Fcfusion protein of SEQ ID NO: 62, containing the cNg-D mutation, exhibitsan Fc (gamma) receptor binding activity that is approximately twice thatof the insulin-Fc fusion proteins of SEQ ID NO: 60, containing the cNg-Smutation and SEQ ID NO: 64 containing the cNg-K mutation. Therefore,only the insulin-Fc fusion proteins comprising the latter two compoundscontaining the cNg-S mutation and the cNg-K mutations were deemedsuitable for repeated dose bioactivity testing in dogs.

TABLE 6 Fc(gamma) receptor binding for cNg variations of SEQ ID NO: 52OD450 nm cNg Log[Fc(gamma) OD450 nm Minus Ratio to SEQ SEQ ID NO:Mutation RI] (ng/mL) Assay Background ID NO: 52 SEQ ID NO: 52 Native cNg0.386 0.323 1.00 SEQ ID NO: 60 cNg-S 0.140 0.077 0.24 SEQ ID NO: 62cNg-D 0.204 0.141 0.44 SEQ ID NO: 64 cNg-K 0.126 0.063 0.20 Assaybackground N/A 0.063 0.000 N/A (no compound)

Example 23: Evaluation of In Vivo Bioactivity and Immunogenicity of anInsulin Polypeptide of SEQ ID NO: 5 with the Non-Glycosylated cNg-K andcNg-S Canine IgGB Isotype Fc Fragments

To determine if the insulin-Fc fusion protein of SEQ ID NO: 60,containing the cNg-S mutation, improved the repeated dose bioactivityperformance in dogs, the compound was administered subcutaneously to N=1dog on day 0, day 7, day 14, and on day 28 according to the procedure ofExample 11. When the dog's % FBGL dropped too low, the dog was givenfood to raise the blood glucose to a safe level. The NAOC for the firstinjection was 191% FBGL·days·kg/mg, showing that the insulin-Fc fusionprotein of SEQ ID NO: 60 was satisfactorily bioactive in vivo. The NAOCand NAOCR were also measured for each subsequent dose according to thegeneral procedure of Example 11, calculated from the time the dose wasadministered until just before the next dose was administered. The NAOCand the NAOCR shown in Table 7 illustrate that the insulin-Fc fusionprotein of SEQ ID NO: 60 exhibited an NAOCR that decreased significantlyon doses 3 and 4 of a four dose regimen. Therefore, the insulin-Fcfusion protein of SEQ ID NO: 60, containing the cNg-S mutation, wasunable to demonstrate repeated dose bioactivity in dogs despite having alow Fc(gamma)RI binding four times lower than that of the insulin-Fcfusion protein of SEQ ID NO: 52.

TABLE 7 NAOC per dose for repeated doses of SEQ ID NO: 60 InjectionNumber of NAOC SEQ ID NO: 60 (% FBGL · days · kg/mg) NAOCR 1 191 1.0 2240 1.3 3 0 0.0 4 39 0.2

To determine if the insulin-Fc fusion protein of SEQ ID NO: 64,containing the cNg-K mutation, improved the repeated dose bioactivityperformance in dogs, the compound was administered subcutaneously to N=1dog on day 0, day 7, day 14, and on day 28 according to the procedure ofExample 11. When the dog's % FBGL dropped too low, the dog was givenfood to raise the blood glucose to a safe level. The NAOC for the firstinjection was 449% FBGL·days·kg/mg, showing that the insulin-Fc fusionprotein of SEQ ID NO: 64 was satisfactorily bioactive in vivo. Thepharmacokinetic profile of the compound was also measured by the methodof Example 12 using ELISA, and a two-compartment model was fit to thedata to determine its elimination half-life which was about 0.9 days.The NAOC and NAOCR were also measured for each subsequent dose accordingto the general procedure of Example 11, calculated from the time thedose was administered until just before the next dose was administered.The NAOC and the NAOCR shown in Table 8 illustrate that the insulin-Fcfusion protein of SEQ ID NO: 64 maintains an NAOCR greater than 0.6throughout the four doses. Therefore, unexpectedly, the insulin-Fcfusion protein of SEQ ID NO: 64, containing the cNg-K mutation, was theonly non-glycosylated mutant of the insulin-Fc fusion protein of SEQ IDNO: 52 resulting in significantly improved repeated dose bioactivity indogs.

TABLE 8 NAOC per dose for repeated doses of SEQ ID NO: 64 InjectionNumber of NAOC SEQ ID NO: 64 (% FBGL · days · kg/mg) NAOCR 1 449 1.0 2361 0.8 3 259 0.6 4 638 1.4

The levels of anti-drug and anti-insulin antibodies were also measuredthroughout the course of treatment (28 days) and for an additional twoweeks according to Example 13. FIG. 9 illustrates that the insulin-Fcfusion protein of SEQ ID NO: 64 still generated anti-drug antibodieswith repeated subcutaneous dosing in dogs, but the anti-drug antibodytiters were much lower than those generated by the insulin-Fc fusionprotein of SEQ ID NO: 52 (Example 19).

Example 24: Screening of Canine Serum Containing Anti-Drug Antibodiesand Identification of Potential Immunogenic Epitopes at the B10D and A8HPositions of the Insulin Polypeptide

Mutating the cNg site of the canine IgGB Fc fragment to a Lys (i.e.,cNg-K) did improve the repeated dose bioactivity of the insulin-fusionprotein comprising the insulin polypeptide of SEQ ID NO: 5 and thepeptide linker of SEQ ID NO: 12 (Example 23), but the resultinginsulin-Fc fusion protein of SEQ ID NO: 64 still gave rise to anti-drugantibodies (Example 23). It was hypothesized, therefore, that theinsulin polypeptide of SEQ ID NO: 5 may unexpectedly contain specificepitopes (i.e., immunogenic “hot spots”) against which the dog's immunesystem is directed. Therefore, the binding specificity of the antibodiespresent in the serum samples described in Example 13 were evaluatedaccording to the general procedure of Example 15. The analysis of theantibody-containing serum samples from the repeated dosing of theinsulin-Fc fusion protein of SEQ ID NO: 52 (Example 19) against thecoated insulin-Fc fusion protein library demonstrated that there wereunexpectedly two primary “hot spots” present within the insulinpolypeptide sequence of SEQ ID NO: 5: the aspartic acid mutation at the10th position from the N-terminus of the B-chain (i.e., B10), and,separately, the histidine mutation at the 8th position from theN-terminal end of the A-chain (i.e., A8). The results suggest thatinsulin-Fc fusion proteins comprising insulin polypeptide amino acidcompositions containing these two particular amino acid mutations arelikely to be immunogenic in dogs and therefore likely to give rise toanti-drug antibodies that neutralize the bioactivity after repeatedinjections. Therefore, it was determined that insulin polypeptides thatdo not contain the B10 aspartic acid and A8 histidine are preferred forinsulin-Fc fusion proteins that need to be repeatedly dosed in dogs overlong periods long-term (e.g., to treat canine diabetes).

Example 25: An Insulin-Fc Fusion Protein Comprising the InsulinPolypeptide of SEQ ID NO: 5 and a Non-Glycosylated Canine IgGB IsotypeFc Fragment in which the B10D and A8H Mutations of the InsulinPolypeptide are Restored to Native Compositions to Reduce the PotentialRisk of Immunogenicity

To evaluate whether replacing the “hot spot” mutations would improve theimmunogenicity and repeated dose bioactivity of insulin-Fc fusionproteins comprising the insulin polypeptide of SEQ ID NO: 5 and thecanine IgGB isotype fragment, an exemplary insulin-Fc fusion protein(SEQ ID NO: 66) was synthesized in which the B10 and A8 amino acids ofthe insulin polypeptide were restored to their native histidine andthreonine compositions, respectively (SEQ ID NO: 125) listed below withnon-native amino acids underlined).

(SEQ ID NO: 125) FVNQHLCGSHLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCTSICSLYQLENYCNFurthermore, given the additional potential benefits of thenon-glycosylated cNg mutants, the insulin-Fc fusion protein of SEQ IDNO: 66 contains the cNg-Q mutation. The entire amino acid sequence ofthe insulin-Fc fusion protein of SEQ ID NO: 66 is given below:

(SEQ ID NO: 66) FVNQHLCGSHLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCTSICSLYQLENYCNGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFQGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

The insulin-Fc fusion protein of SEQ ID NO: 66 was manufactured inHEK293 cells according to Example 1 and purified using a Protein Acolumn according to Example 3. The resulting protein yield was only 21mg/L. The structure was confirmed according to Example 4 by non-reducingand reducing CE-SDS, and the sequence was further identified by LC-MSwith glycan removal according to Example 5. The % homodimer as measuredby size-exclusion chromatography according to Example 6, was 98.0%indicating that the protein was relatively free of aggregates.

Despite the relatively low homodimer titer of 21 mg/L, the insulin-Fcfusion protein of SEQ ID NO: 66 was evaluated in dogs for in vivobioactivity and immunogenicity according to the procedures of Examples11-13, respectively. FIG. 10 demonstrates that restoration of the B10Dand A8H mutations to their native amino acids (i.e., B10H and A8T) inthe insulin-Fc fusion protein of SEQ ID NO: 66 did significantly reducethe immunogenicity of the parent compound (SEQ ID NO: 52).

However, as shown in FIG. 11, the insulin-Fc fusion protein of SEQ IDNO: 66 containing the native B10 and A8 amino acids was not bioactive(i.e., the NAOC was essentially zero).

Example 26: Attempts to Incorporate Additional B-Chain and A-ChainMutations into the Insulin Polypeptide of SEQ ID NO: 125 to Improve theBioactivity of the Associated Insulin-Fc Fusion Proteins Containing theCanine IgGB Fc Fragment

The fact that the insulin-Fc fusion protein of SEQ ID NO: 66 did notgenerate anti-drug antibodies (Example 25) compared to the insulin-Fcfusion protein of SEQ ID NO: 52 (Example 20) provides strong evidencefor the theory that the B10D and A8H mutations in the insulinpolypeptide of SEQ ID NO: 5 are likely the immunogenic epitopesresponsible for the production of anti-drug antibodies. However, thelack of in vivo potency of the insulin-Fc fusion protein of SEQ ID NO:66 compared to that of SEQ ID NO: 52 indicates that these two amino acidmutations are also responsible for achieving acceptable levels ofbioactivity. The lack of in vivo potency for the insulin-Fc fusionprotein of SEQ ID NO: 66 correlates with its high IC50 (shown in Table 9below) as measured by the insulin receptor binding assay according tothe method of Example 7. Therefore, further efforts were required toincrease the insulin-Fc fusion protein bioactivity (i.e., decrease theinsulin receptor binding assay IC50 value to less than 5000 nM, or morepreferably less than 4000 nM, or even more preferably less than 3000 nM)while maintaining a low degree of immunogenicity by keeping the nativeB10 and A8 amino acids in the insulin polypeptide.

It is known that various portions of the insulin B-chain and A-chain arerequired for strong binding to the IR (Hubbard S. R., “Structuralbiology: Insulin meets its receptor”, Nature. 2013; 493(7431):171-172).Therefore, portions of the B-chain or A-chain were modified whilekeeping the B10 and A8 the same as in native insulin and the C-chain andpeptide linker constant. Several of these insulin-Fc fusion proteinswere manufactured in HEK293 cells according to Example 1 and purifiedusing a Protein A column according to Example 3. Their structures wereconfirmed according to Example 4 by non-reducing and reducing CE-SDS,and the sequences were further identified by LC-MS with glycan removalaccording to Example 5. Their % homodimer content was measured bysize-exclusion chromatography according to Example 6, and their insulinreceptor binding affinities were measured according to Example 7. Theirsequences are shown below, and the resulting sequence alignments againstSEQ ID NO: 66 are shown in FIG. 12 (Clustal Omega).

(SEQ ID NO: 68) FVNQHLCGSHLVQALYLVCGERGFFYTDPTGGGPRRGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 70)FVNQHLCGSELVEALALVCGERGFFYTDPTGGGPRRGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 72)FVNQHLCGSHLVEALALVCGEAGFFYTDPTGGGPRRGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 74)FVNQHLCGSHLVEALALVCGERGFYYTDPTGGGPRRGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 76)FVNQHLCGSHLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

TABLE 9 % homodimer, homodimer titers, and IR IC50 values for variousSEQ ID NOs. HEK homodimer titer IR IC50 SEQ ID NO: % Homodimer (mg/L)(nM) SEQ ID NO: 66 98.0% 21 >5000 SEQ ID NO: 68 97.6% 9 2624 SEQ ID NO:70 81.4% 17 633 SEQ ID NO: 72 99.1% 22 >5000 SEQ ID NO: 74 96.6% 25 2402SEQ ID NO: 76 98.0% 6 >5000

In only three cases (SEQ ID NOs: 68, 70, and 74 did the proposedmutations improve the IR binding (i.e., lower the IC50 value) ascompared to SEQ ID NO: 66. However, none of the mutations resulted incompounds that meet the manufacturing design goal of a homodimer titergreater than 50 mg/L, and in some cases, the mutations lead tosignificantly reduced manufacturability (e.g., homodimer titers lessthan 20 mg/L).

Example 27: Attempts to Incorporate C-Chain Mutations into the InsulinPolypeptide of SEQ ID NO: 125 to Improve the Bioactivity of theAssociated Insulin-Fc Fusion Proteins Containing the Canine IgGB FcFragment

The results obtained in Example 26 showed that all attempts to mutatethe A-chain and B-chain of the insulin polypeptide of SEQ ID NO: 125resulted in unacceptably low HEK homodimer titers of the associatedinsulin-Fc fusion (i.e., homodimer titers less than or equal to 25mg/L). Therefore, there was a need for further experimentation. In thepresent example, the C-chain composition of the insulin polypeptide ofSEQ ID NO: 125 was mutated by making it longer or by increasing itsflexibility. Native insulin (e.g. human insulin) has been shown toundergo a significant conformational change that involves movement ofboth the B-chain and A-chain folding as it binds to the insulin receptor(e.g., as described by Menting, et al., Nature, 2013; 493(7431): pp241-245). Native insulin, unlike the insulin polypeptides of the presentinvention, is freely able to undergo this conformational change at theinsulin receptor, because it is a two-chain polypeptide in its nativeform, connected only through two disulfide bonds with no C-chainconstraining the mobility of the A- and B-chains. Without being bound byany particular theory, it was hypothesized that the C-chain containedwithin the insulin polypeptide of SEQ ID NO: 125 was too inflexible(e.g. an amino acid composition and sequence that does not permit facilemovement between the B-chain and A-chain) and/or too short (e.g. notenough amino acids between the C-terminus of the B-chain and theN-terminus of the A-chain) thus preventing the insulin polypeptide fromundergoing the necessary change in molecular shape required for strongbinding to the insulin receptor. Therefore, several insulin-Fc fusionproteins were synthesized based on the insulin-Fc fusion protein of SEQID NO: 66 with variations in the insulin polypeptide C-chain as shownbelow with the resulting sequence alignments against SEQ ID NO: 66 shownin FIG. 13 (Clustal Omega).

(SEQ ID NO: 78) FVNQHLCGSHLVQALYLVCGERGFFYTDPTQRGGGGGQRGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSK LSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 80)FVNQHLCGSHLVEALALVCGERGFFYTDPTGGGGGGSGGGGGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 82)FVNQHLCGSHLVEALALVCGERGFFYTDPGGGGGGGGGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 84)FVNQHLCGSHLVEALALVCGERGFFYTPGGGGGGGGGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

TABLE 10 % homodimer, homodimer titers, and IR IC50 values for variousSEQ ID NOs. HEK homodimer titer IR IC50 SEQ ID NO: % Homodimer (mg/L)(nM) SEQ ID NO: 66 98.0% 21 >5000 SEQ ID NO: 78 94.0% 8 4176 SEQ ID NO:80 99.6% 37 1609 SEQ ID NO: 82 98.3% 42 >5000 SEQ ID NO: 84 98.6% 334720

The insulin-Fc fusion proteins were manufactured in HEK293 cellsaccording to Example 1 and purified using a Protein A column accordingto Example 3. Their structures were confirmed according to Example 4 bynon-reducing and reducing CE-SDS, and the sequences were furtheridentified by LC-MS with glycan removal according to Example 5. Their %homodimer content was measured by size-exclusion chromatographyaccording to Example 6, and their insulin receptor binding affinitieswere measured according to Example 7. In only one case, (SEQ ID NO: 80)which comprises the longest C-chain (GGGGGGSGGGG—SEQ ID NO: 133), did aC-chain mutation significantly improve the insulin receptor bindingaffinity (IC50 less than 3000 nM) compared to that of the insulin-Fcfusion protein of SEQ ID NO: 66. However, none of these C-chain-mutatedinsulin-Fc fusion proteins exhibited a homodimer titer greater than themanufacturing design goal of 50 mg/L. In fact, in one case (SEQ ID NO:78) the C-chain mutation unexpectedly led to significantly lowerhomodimer titers.

Example 28: Attempts to Incorporate Peptide Linker Mutations intoInsulin-Fc Fusion Proteins Containing the Insulin Polypeptide of SEQ IDNO: 125 and the Canine IgGB Fc Fragment to Improve Bioactivity

Without being bound by any particular theory, another possible reasonfor the poor insulin receptor binding of the insulin-Fc fusion proteinof SEQ ID NO: 66 was thought to involve the steric hindrance between theinsulin polypeptide and the insulin receptor resulting from the closeproximity of the much larger Fc fragment molecule attached to theinsulin polypeptide through the peptide linker. Shorter peptide linkersor more tightly folded peptide linkers were thought to potentiallyexacerbate this issue, while longer peptide linkers or peptide linkersthat are resistant to folding back on themselves (e.g., linkers withmore molecular stiffness) may alleviate this issue by creating morespace between the insulin polypeptide and the Fc fragment. The increasedspace between the insulin polypeptide and the Fc fragment would alsoincrease the distance between the insulin receptor and the Fc fragmentleading to less interference during insulin receptor binding. Thepeptide linker of SEQ ID NO: 12 (i.e., GGGGAGGGG) used to construct theinsulin-Fc fusion protein of SEQ ID NO: 66 was hypothesized to bepotentially too short and/or too flexible, because the amino acids thatcomprise the linker contain no side chains (i.e., it contains onlyglycine and alanine amino acids). Therefore, to test this hypothesis,two other insulin-Fc fusion protein variants of the insulin-Fc fusionprotein of SEQ ID NO: 66 were synthesized. The insulin-Fc fusion proteinof SEQ ID NO: 76 contained the same peptide linker as was used toconstruct the insulin-Fc fusion protein of SEQ ID NO: 66 but with aninsulin polypeptide in which the asparagine at the 21^(st) position fromthe N-terminus of the A chain (i.e., A21) was absent (i.e., des-A21).This particular mutation was incorporated to see whether the junctionbetween the A-chain and the peptide linker affects the protein yieldand/or bioactivity of the molecule. The other insulin-Fc fusion proteinof SEQ ID NO: 86 contains this des-A21N A-chain mutation and a peptidelinker that is more than twice the length of that used to construct theinsulin-Fc fusion protein of SEQ ID NO: 66. In this longer peptidelinker, alanine is disfavored and instead is replaced with a glutamine,which contains a polar amide side chain. The glutamine substitutionswere expected to increase the hydrophilic nature of the peptide linkerand potentially prevent the linker from folding back against itself. Thesequences are shown below with the resulting sequence alignments againstSEQ ID NO: 66 shown in FIG. 14 (Clustal Omega).

(SEQ ID NO: 86) FVNQHLCGSHLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCTSICSLYQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLD EDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 76)FVNQHLCGSHLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

TABLE 11 % homodimer, homodimer titers, and IR IC50 values for variousSEQ ID NOs. HEK Homodimer titer IR IC50 SEQ ID NO: % Homodimer (mg/L)(nM) SEQ ID NO: 66 98.0% 21 >5000 SEQ ID NO: 76 98.0% 6 >5000 SEQ ID NO:86 99.6% 11 1281

The two insulin-Fc fusion proteins were manufactured in HEK293 cellsaccording to Example 1 and purified using a Protein A column accordingto Example 3. Their structures were confirmed according to Example 4 bynon-reducing and reducing CE-SDS, and the sequences were furtheridentified by LC-MS with glycan removal according to Example 5. Their %homodimer content was measured by size-exclusion chromatographyaccording to Example 6, and their insulin receptor binding affinitieswere measured according to Example 7. The incorporation of a longerpeptide linker of different composition (GGGGGQGGGGQGGGGQGGGGG (SEQ IDNO: 14) for SEQ ID NO: 86 vs. GGGGAGGGG (SEQ ID NO: 12) for SEQ ID NO:66) did improve the insulin receptor binding as measured by asignificant reduction in the IC50 value, indicating that longer linkersmay be a strategy for increasing insulin receptor binding for otherinsulin-Fc fusion proteins. However, the incorporation of a longerlinker still did not improve the homodimer titers to above themanufacturing design goal of greater than 50 mg/L.

Example 29: Attempts to Delete Portions of the B-Chain of the InsulinPolypeptide of SEQ ID NO: 125 to Improve the Homodimer Titer of theAssociated Insulin-Fc Fusion Proteins Containing the Canine IgGB FcFragment

The results from Example 28 demonstrate that the peptide linker can bemodified to increase the insulin receptor binding affinity of theinsulin-Fc fusion protein of SEQ ID NO: 66, which contains the nativeB10 and A8 amino acids. However, the peptide linker mutation failed toincrease the homodimer titer enough to meet the manufacturing designgoal. Because the homodimer titer is a function of several properties,including the intracellular synthesis and processing within cells, itwas hypothesized that perhaps the insulin-Fc molecule wasself-associating (i.e., aggregating) during and after synthesis eitherintramolecularly between the two monomers of the homodimer orintermolecularly between two or more separate homodimers. Thisaggregation would lead to unacceptably low homodimer titers obtainedfrom the cell culture supernatants during the production processdescribed in Examples 1, 3, and 6. This potential interaction betweenthe insulin-Fc fusion protein molecules could be due, in part, toinsulin's well-known propensity to self-associate and form aggregates.One method known in the art to reduce the propensity for insulin toself-associate involves mutating the amino acids near the C-terminus ofthe B-chain. For example, insulin lispro (B28K; B29P mutations) andinsulin aspart (B28D mutation) are well-known commercial two-chaininsulins with non-native B-chain mutations that prevent association andaggregation thus giving rise to a predominantly monomeric form ofinsulin in solution. Another approach to prevent aggregation involvesamino acid structural deletions. For example, a two-chain insulin knownas despentapeptide insulin (DPPI; see Brange J., Dodson G. G., EdwardsJ., Holden P. H., Whittingham J L 1997b. “A model of insulin fibrilsderived from the x-ray crystal structure of a monomeric insulin(despentapeptide insulin)” Proteins 27 507-516), is identical to nativetwo-chain human insulin except that the five C-terminal amino acids ofthe B-chain (YTPKT) are removed. DPPI has a lower binding affinity tothe insulin receptor as compared to the native two-chain human insulin,but it is completely monomeric in solution, meaning that there is nosignificant association or aggregation between DPPI molecules.Therefore, in an attempt to decrease the potential for intramolecularand intermolecular self-association and improve the insulin-Fc fusionprotein homodimer titer, several variants of the insulin-Fc fusionprotein of SEQ ID NO: 66 were constructed using partial B-chain aminoacid truncation and B-chain amino acid mutations as described above forDPPI, insulin lispro, and insulin aspart. The sequences are shown belowwith the resulting sequence alignments against SEQ ID NO: 66 shown inFIG. 15 (Clustal Omega).

(SEQ ID NO: 82) FVNQHLCGSHLVEALALVCGERGFFYTDPGGGGGGGGGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 84)FVNQHLCGSHLVEALALVCGERGFFYTPGGGGGGGGGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 88)FVNQHLCGSHLVEALALVCGERGFFYTQGGGGGGGGGIVEQCCTSICSLYQLENYCGGGGAGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

TABLE 12 % homodimer, homodimer titers, and IR IC50 values for variousSEQ ID NOs. HEK Homodimer titer IR IC50 SEQ ID NO: % Homodimer (mg/L)(nM) SEQ ID NO: 66 98.0% 21 >5000 SEQ ID NO: 82 98.3% 42 1915 SEQ ID NO:88 99.4% 22 2195 SEQ ID NO: 84 98.6% 33 1930

The insulin-Fc fusion proteins were manufactured in HEK293 cellsaccording to Example 1 and purified using a Protein A column accordingto Example 3. Their structures were confirmed according to Example 4 bynon-reducing and reducing CE-SDS, and the sequences were furtheridentified by LC-MS with glycan removal according to Example 5. Their %homodimer content was measured by size-exclusion chromatographyaccording to Example 6, and their insulin receptor binding affinitieswere measured according to Example 7. The homodimer titer of theresulting compounds was only significantly increased in one case (SEQ IDNO: 82), but unexpectedly, the insulin receptor affinity was improvedfor all of the mutated compounds (SEQ ID NOs: 82, 88, and 84).

Example 30: Attempts to Combine B-Chain, C-Chain, and A-Chain Mutations,B-Chain Truncation, and Linker Mutations to the Insulin-Fc FusionProtein of SEQ ID NO: 66 to Further Improve Homodimer Titer andBioactivity

As shown in Examples 26, 27, 28, and 29, no single strategy successfullyincorporated an insulin polypeptide comprising the non-immunogenicnative B10 and A8 amino acids with the canine IgGB Fc fragment to forman insulin-Fc fusion protein with acceptable insulin receptor activityand homodimer titer. Therefore, the concepts of a longer C-chain, alonger peptide linker, and truncation of the C-terminal amino acids ofthe B-chain were combined. In addition, to potentially further decreasethe propensity for self-association and aggregation, additional pointmutations were introduced to the native insulin hydrophobic amino acidresidue sites using less hydrophobic amino acids, including those withside groups that are negatively or positively charged at physiologicalpH. Example mutations included tyrosine to alanine, tyrosine to glutamicacid, isoleucine to threonine, and phenylalanine to histidine.Furthermore, to simplify the analysis, in all cases the cNg site of thecanine IgGB Fc fragment was restored to its native asparagine. Thesequences for these insulin-Fc fusion protein variants are shown belowwith the resulting sequence alignments against SEQ ID NO: 66 shown inFIG. 16 (Clustal Omega).

(SEQ ID NO: 90) FVNQHLCGSHLVEALELVCGERGFFYTPKTGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 92)FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCNHGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 34)FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCNGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQ LDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 32)FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 94)FVNQHLCGSHLVEALELVCGERGFFYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

TABLE 13 % homodimer, homodimer titers, and IR IC50 values for variousSEQ ID NOs. HEK homodimer titer IR IC50 SEQ ID NO: % Homodimer (mg/L)(nM) SEQ ID NO: 66 98.0% 21 >5000 SEQ ID NO: 90 97.9% 69 3869 SEQ ID NO:92 99.5% 101 554 SEQ ID NO: 34 99.7% 107 1247 SEQ ID NO: 94 99.7% 1282043 SEQ ID NO: 32 99.4% 187 2339

The insulin-Fc fusion proteins were manufactured in HEK293 cellsaccording to Example 1 and purified using a Protein A column accordingto Example 3. Their structures were confirmed according to Example 4 bynon-reducing and reducing CE-SDS, and the sequences were furtheridentified by LC-MS with glycan removal according to Example 5. Their %homodimer content was measured by size-exclusion chromatographyaccording to Example 6, and their insulin receptor binding affinitieswere measured according to Example 7. The results show that acombination of decreasing the hydrophobicity of certain B-chain andA-chain amino acids, using longer and more flexible C-peptide sequences,truncating several C-terminal B-chain amino acids, and using a longerpeptide linker resulted in several useful insulin-Fc fusion proteinsthat meet the minimum homodimer titer and insulin receptor bindingactivity design criteria. SEQ ID NOs: 92, 34, 32, and 94 (368d), (366d),(218d), and (375d) showed more preferable insulin receptor IC50 values(less than 3000 nM) and more preferable HEK homodimer titer values(greater than 100 mg/L) than either SEQ ID NO: 66 or SEQ ID NO: 90.Surprisingly, changing just a few amino acids leads to a multifoldimprovement in insulin receptor affinity, and, in the case of theinsulin-Fc fusion protein of SEQ ID NO: 32 a dramatic increase inhomodimer titer over the original insulin-Fc fusion protein of SEQ IDNO: 66.

Example 31: In Vivo Bioactivity, Repeated Dose Bioactivity, andImmunogenicity of Insulin-Fc Fusion Proteins Constructed from theInsulin Polypeptide of SEQ ID NO: 7, the Peptide Linker of SEQ ID NO:14, and the Canine IgGB Fc Fragment of SEQ ID NO: 16

Given the positive homodimer titer and insulin receptor binding activityresults from Example 30, two of the most promising insulin-Fc fusionproteins (SEQ ID NOs: 32 and 34) were tested in dogs to evaluate therepeated dose bioactivity and immunogenicity. Each compound comprisesthe longer, more hydrophilic peptide linker of SEQ ID NO: 14 and themore manufacturable, less aggregated canine IgGB Fc fragment of SEQ IDNO: 16. Most importantly, both insulin-Fc fusion proteins compriseinsulin polypeptides with the putatively less immunogenic native B10 andA8 amino acids (i.e. general SEQ ID NO: 7). In the case of theinsulin-Fc fusion protein of SEQ ID NO: 34, the asparagine at positionA21 is present (i.e. the insulin polypeptide comprises SEQ ID NO: 9). Inthe case of the insulin-Fc fusion protein of SEQ ID NO: 32, theasparagine at position A21 is absent (i.e. the insulin polypeptidecomprises SEQ ID NO: 8).

The in vivo bioactivity of the insulin-Fc fusion protein of SEQ ID NO:34 was tested in N=1 dog according to the procedure of Example 10. Theresults shown in FIG. 17 for a single subcutaneous dose demonstrate thatthe insulin-Fc fusion protein of SEQ ID NO: 34 is indeed bioactive invivo with an NAOC of 1076% FBGL·days·kg/mg calculated according to theprocedure in Example 11. The insulin-Fc fusion protein of SEQ ID NO: 34pharmacokinetic profile was measured by the method of Example 12 usingELISA, and a two-compartment model was fit to the data to determine itselimination half-life which was 3.5 days.

The repeated dose bioactivity was then evaluated by continuing tosubcutaneously administer the insulin-Fc fusion protein of SEQ ID NO: 34to N=1 dog on day 14, day 28, and day 42 after the initial injectionaccording to the procedure of Example 8. When the dog's % FBGL droppedtoo low, the dog was given food to raise the blood glucose to a safelevel. The NAOC and NAOCR were measured for each subsequent doseaccording to the general procedure of Example 11, calculated from thetime the dose was administered until just before the next dose wasadministered. The NAOC and the NAOCR shown in Table 14 illustrate thatthe insulin-Fc fusion protein of SEQ ID NO: 34 maintains an NAOCRgreater than 0.8 throughout the four doses thus meeting the repeateddose bioactivity design goal.

TABLE: 14 NAOC per dose for repeated doses of SEQ ID NO: 34 NAOCInjection# Day (% FBGL · days · kg/mg) NAOCR 1 0 1076 1.0 2 14 1005 0.93 28 900 0.8 4 42 838 0.8

The immunogenicity of the insulin-Fc fusion protein of SEQ ID NO: 34 wastested according to the procedure of Example 13. FIG. 18 demonstratesthat the insulin-Fc fusion protein of SEQ ID NO: 34 exhibits no apparentimmunogenicity in vivo in agreement with the maintenance of in vivobioactivity throughout the repeated dose experiment.

The insulin-Fc fusion protein of SEQ ID NO: 32, with the asparagine atA21 of the insulin polypeptide chain absent, was also evaluated forrepeated dose bioactivity performance in dogs. The compound wasadministered subcutaneously to N=1 dog on day 0, day 14, day 28, and onday 42 according to the procedure of Example 11. When the dog's % FBGLdropped too low, the dog was given food to raise the blood glucose to asafe level. The NAOC for the first injection was an impressive 2278%FBGL·days·kg/mg, showing that the insulin-Fc fusion protein of SEQ IDNO: 32 was satisfactorily bioactive in vivo at almost twice the potencyof the insulin-Fc fusion protein of SEQ ID NO: 34. The pharmacokineticprofile of the insulin-Fc fusion protein was measured by the method ofExample 12 using ELISA, and a two-compartment model was fit to the datato determine its elimination half-life which was 4.1±0.7 days. FIGS. 19and 20 show the single dose blood glucose control and the multidose,multiweek blood glucose control for animals receiving the homodimer ofSEQ ID NO: 32. The NAOC and NAOCR were also measured for each subsequentdose according to the general procedure of Example 11, calculated fromthe time the dose was administered until just before the next dose wasadministered. The NAOC and the NAOCR shown in Table 15 illustrate thatthe insulin-Fc fusion protein of SEQ ID NO: 32 maintains an NAOCRgreater than or equal to 1.0 throughout the four doses thus meeting therepeated dose bioactivity design goal described in Example 16.

The immunogenicity of the insulin-Fc fusion protein of SEQ ID NO: 32 wastested according to the procedure of Example 13. FIG. 21 demonstratesthat the insulin-Fc fusion protein of SEQ ID NO: 32 exhibits no apparentimmunogenicity in vivo in agreement with the maintenance of in vivobioactivity throughout the repeated dose experiment.

TABLE 15 NAOC per dose for repeated doses of SEQ ID NO: 32 NAOCInjection# Day (% FBGL · days · kg/mg) NAOCR 1 0 2278 1.0 2 14 4029 1.83 28 3450 1.5 4 42 3257 1.4

As discussed in the Detailed Description of the invention, a knownenzymatic cleavage site exists between asparagine-glycine bonds (Vlasak,J., Ionescu, R., (2011) MAbs Vol. 3, No. 3 pp 253-263). Omitting theasparagine at the 21st amino acid in the A chain (i.e., A21) in theinsulin polypeptide of SEQ ID NO: 8 contained in the insulin-Fc fusionprotein of SEQ ID NO: 32 with the peptide linker of SEQ ID NO: 14,eliminates the possibility of enzymatic cleavage of theasparagine-glycine bond between the C-terminus of the A-chain and theN-terminus of the peptide linker. However, the insulin-Fc fusion proteinof SEQ ID NO: 34 comprises the peptide linker of SEQ ID NO: 14 and theinsulin polypeptide of SEQ ID NO: 8, which keeps the asparagine at A21.Therefore, it would have been expected that the insulin-Fc fusionprotein of SEQ ID NO: 34 would have been enzymatically digested duringsynthesis or in vivo following subcutaneous administration. However,rather unexpectedly the insulin-Fc fusion protein of SEQ ID NO: 34 wasmanufacturable in HEK cells with an acceptable homodimer titer anddemonstrated acceptable bioactivity in vivo with no signs of enzymaticdigestion compromising its bioactivity.

Example 32: Confirmation of the Canine IgGB Isotype Fc Fragment forOptimal Manufacturability and In Vivo Efficacy of Insulin-Fc FusionProteins Comprising the Preferred Insulin Polypeptide of SEQ ID NO: 8and the Preferred Peptide Linker of SEQ ID NO: 14

Having discovered a new insulin polypeptide and peptide linkercombination resulting in non-immunogenic, high yielding, high purity,and highly bioactive insulin-Fc fusion proteins as described in Examples30 and 31, a question remained as to whether the canine IgGB Fc fragmentwas still the preferred isotype with respect to homodimer titer andbioactivity as was the case for the insulin-Fc fusion proteins inExamples 19 and 20. Therefore, additional insulin-Fc fusion proteinswere designed wherein the insulin polypeptide (SEQ ID NO: 8) and peptidelinker (SEQ ID NO: 14) of the insulin-Fc fusion protein of SEQ ID NO: 32were kept constant, and the canine IgGB Fc fragment of SEQ ID NO: 16 wasreplaced by the canine IgGA Fc fragment of SEQ ID NO: 15, the canineIgGC Fc fragment of SEQ ID NO: 17, or the canine IgGD Fc fragment of SEQID NO: 18. The sequences for these resulting insulin-Fc fusion proteinvariants are shown below:

(SEQ ID NO: 32) FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 96)FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSPG (SEQ ID NO: 98)FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGCNNCPCPGCGLLGGPSVFIFPPKPKDILVTARTPTVTCVVVDLDPENPEVQISWFVDSKQVQTANTQPREEQSNGTYRVVSVLPIGHQDWLSGKQFKCKVNNKALPSPIEEIISKTPGQAHQPNVYVLPPSRDEMSKNTVTLTCLVKDFFPPEIDVEWQSNGQQEPESKYRMTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQISLSHSPG (SEQ ID NO: 100)FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGCISPCPVPESLGGPSVFIFPPKPKDILRITRTPEITCVVLDLGREDPEVQISWFVDGKEVHTAKTQPREQQFNSTYRVVSVLPIEHQDWLTGKEFKCRVNHIGLPSPIERTISKARGQAHQPSVYVLPPSPKELSSSDTVTLTCLIKDFFPPEIDVEWQSNGQPEPESKYHTTAPQ LDEDGSYFLYSKLSVDKSRWQQGDTFTCAVMHEALQNHYTDLSLSHSPG

The insulin-Fc fusion proteins were manufactured in HEK293 cellsaccording to Example 1 and purified using a Protein A or Protein Gcolumns according to Example 3. Their structures were confirmedaccording to Example 4 by non-reducing and reducing CE-SDS, and thesequences were further identified by LC-MS with glycan removal accordingto Example 5. Their % homodimer content was measured by size-exclusionchromatography according to Example 6, and their insulin receptorbinding affinities were measured according to Example 7. Additionallythe insulin-Fc fusion protein affinities for the canine FcRn receptorwere measured according to Example 8. As is shown in Table 16, theinsulin-Fc fusion protein of SEQ ID NO: 32 comprising the canine IgGB Fcfragment demonstrated the highest homodimer titer of these sequences.The insulin-Fc fusion protein of SEQ ID NO: 96 comprising the canineIgGA Fc fragment exhibited poor homodimer titer when purified using aProtein A column; however, when it purified using a Protein G column,the homodimer titer was significantly improved, exceeding the designgoal of greater than 50 mg/L. The same was true for the insulin-Fcfusion protein of SEQ ID NO: 98 comprising the canine IgGC Fc fragment.The insulin-Fc fusion protein of SEQ ID NO: 100 comprising the canineIgGD Fc fragment did not yield any compound when purified with either aProtein A or a Protein G column. Therefore, as was demonstrated with theinsulin-Fc fusion protein of SEQ ID NO: 52 containing a differentinsulin polypeptide (SEQ ID NO: 5 and peptide linker (SEQ ID NO: 12),the canine IgGB was the preferred Fc fragment with respect to homodimertiter (see Example 19).

TABLE 16 Homodimer titers, IR binding, and FcRn binding for sequencesutilizing native canine IgGA, IgGB, IgGC and IgGD Fc fragments ProteinFc Yield Homo- IR FcRn First dose Fragment Protein A/ % Homo-dimer dimerBinding, Binding, NAOC SEQ ID IgG (Protein G) Protein A/ Titer IC50 EC50(% FBGL · NO: Isotype (mg/L) (Protein G) (mg/L) (nM) (ng/mL) days ·kg/mg) SEQ ID IgGB 187/(DNM) 99%/(DNM) 185 2339     599 2278 NO: 32 SEQID IgGA  10/(69) 45%/(91%)  62^(‡) 2586^(#)    1610  174 NO: 96 SEQ IDIgGC  0/(86)  0%/(94%)  81^(‡) 2084^(‡) >200000  39 NO: 98 SEQ ID IgGD 0/(0) (DNM)/(DNM)  0 DNM DNM DNM NO: 100 DNM = did not measure; ^(#)=purified via Protein A; ^(‡)= purified by Protein G.

The in vivo bioactivity of the insulin-Fc fusion protein of SEQ ID NO:96 comprising the canine IgGA Fc fragment that was purified via ProteinG was tested according to the procedure of Example 10. The resultsillustrated in FIG. 22 show that the insulin-Fc fusion protein of SEQ IDNO: 96 is only somewhat bioactive in vivo with a NAOC of only 174%FBGL·days·kg/mg calculated according to Example 11.

The in vivo bioactivity of the insulin-Fc fusion protein of SEQ ID NO:98 comprising the canine IgGC Fc fragment was purified via Protein Gtested according to the procedure of Example 10. The results illustratedin FIG. 23 show that the insulin-Fc fusion protein of SEQ ID NO: 98 isonly somewhat bioactive in vivo with a NAOC of only 39% FBGL·days·kg/mgcalculated according to Example 11.

Therefore, as was demonstrated with the insulin-Fc fusion protein of SEQID NO: 52 containing a different insulin polypeptide (SEQ ID NO: 5) andpeptide linker (SEQ ID NO: 12), the canine IgGB was the preferred Fcfragment with respect to bioactivity (see Examples 19 and 20 and Table16 above).

Example 33: Non-Glycosylated Insulin-Fc Fusion Proteins Comprising theInsulin Polypeptide of SEQ ID NO: 8, the Peptide Linker of SEQ ID NO:14, and the Canine IgGB Fc Fragment to Reduce the Potential Risk ofImmunogenicity

While the insulin-Fc fusion protein of SEQ ID NO: 32 meets all of thedesign goals (Example 16), there may or may not be a risk ofimmunogenicity over extended periods of treatment (e.g., 6 months, 1year, 2 years or more) which could compromise the use of this insulin-Fcfusion protein for treating diabetes should this occur. As described inthe Detailed Description of the Invention and in Examples 21 and 22, onepossible cause of a reduction in bioactivity after repeated doses is theunwanted interaction of the canine IgGB Fc fragment with the dog'simmune system resulting in the production of neutralizing anti-drugantibodies. However, the results shown in Example 32 demonstrate thatunexpectedly, the canine IgGB isotype was the only option of the fourcanine IgG isotypes that yielded the desired manufacturability andbioactivity. Therefore, further Fc mutations were explored to achievenon-glycosylated insulin-Fc fusion proteins with low Fc(gamma)RIreceptor binding, which should reduce the long-term, chronicimmunogenicity risk.

As described in the Detailed Description of the Invention, one methodfor reducing the Fc(gamma)RI interaction involves mutating the Fcfragment cNg site to prevent glycosylation during synthesis in the hostcell. Therefore, cNg site mutations were made to the Fc fragment regionof SEQ ID NO: 32 to reduce the binding affinity of the Fc fragment forFc(gamma) receptors in vivo, as measured by binding in an in vitro humanFc(gamma)RI assay described in Example 8. The position of the cNg sitein the insulin-Fc fusion protein of SEQ ID NO: 32 is cNg-NB151.Mutations to SEQ ID NO: 32 included SEQ ID NO: 104 comprising acNg-NB151-S mutation and SEQ ID NO: 102 comprising the same cNg-NB151-Smutation as well as a NB119-A mutation. The NB119-A was incorporated ina further attempt to reduce the interaction with Fc(gamma)RI as has beendescribed only for use in mouse antibodies by Lo, M. et al. “Effectorattenuating substitutions that maintain antibody stability and reducetoxicity in mice”, J. Biol. Chem. (2017), pp. 1-20. The full amino acidsequences of the resulting insulin-Fc fusion proteins are listed below(NB119 and NB151 sites underlined for clarity) along with their sequencealignments (Clustal Omega) which are shown in FIG. 24:

(SEQ ID NO: 102) FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG (SEQ ID NO: 104)FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG

The insulin-Fc fusion proteins were manufactured in HEK293 cellsaccording to Example 1 and purified using a Protein A column accordingto Example 3. Their structures were confirmed according to Example 4 bynon-reducing and reducing CE-SDS, and the sequences were furtheridentified by LC-MS with glycan removal according to Example 5. Their %homodimer content was measured by size-exclusion chromatographyaccording to Example 6, and their insulin receptor binding affinitieswere measured according to Example 7. As shown in Table 17,incorporating the cNg-NB151-S mutations on the Fc fragment decreased the% homodimer, indicating an unacceptably high level of aggregation (i.e.,the % homodimer dropped to just above 70%).

TABLE 17 Homodimer titers for non-glycosylated insulin-Fc fusionproteins of SEQ ID NO: 102 and 104 Homo- IR IgG Protein % dimer Binding,SEQ ID Frag- Relevant Yield Homo- Titer IC50 NO: ment Mutations (mg/L)dimer (mg/L) (nM) SEQ ID IgGB cNg-NB-151-N 187 99% 185 2339 NO: 32 SEQID IgGB cNg-NB-151-S, 78 73% 57 3093 NO: 102 NB119-A SEQ ID IgGBcNg-NB151-S 130 71% 93 2302 NO: 104

The in vivo bioactivity of the insulin-Fc fusion proteins of SEQ ID NO:102 and SEQ ID NO: 104 were tested in N=1 dog each according to theprocedure of Example 10. The results shown in FIG. 25 for a singlesubcutaneous dose demonstrate that both compounds were significantlyless bioactive in vivo than the insulin-Fc fusion protein of SEQ ID NO:32 (NAOC for SEQ ID NO: 104=574% FBGL·days·kg/mg; NAOC for SEQ ID NO:102=921% FBGL·days·kg/mg). The results indicate that incorporatingcNg-NB151-S mutations on the Fc fragment to produce non-glycosylatedversions of the insulin-Fc fusion protein of SEQ ID NO: 32 unexpectedlydecreased the in vivo bioactivity of the resulting compounds.

In an attempt to lessen the degree of aggregation and improve thebioactivity of the insulin-Fc fusion protein of SEQ ID NO: 104containing the cNg-NB151-S site mutation, various insulin-polypeptideB-chain variants were investigated with mutations in the region thoughtto be responsible for aggregation. The insulin-Fc fusion proteins weremanufactured in HEK293 cells according to Example 1 and purified using aProtein A column according to Example 3. Their structures were confirmedaccording to Example 4 by non-reducing and reducing CE-SDS, and thesequences were further identified by LC-MS with glycan removal accordingto Example 5. Their % homodimer content was measured by size-exclusionchromatography according to Example 6. Among the B-chain variantstested, one insulin Fc-fusion protein (SEQ ID NO: 36) containing atyrosine to alanine substitution at the 16^(th) amino acid from theN-terminus of the B-chain (i.e., B16) was unexpectedly found to havehigh homodimer titers (105 mg/L) with low aggregation (99% homodimer),resulting in a homodimer titer of 104 mg/L. The insulin receptor bindingmeasured according to Example 7 was acceptable with an IC50 of 2040 nM.The FcRn receptor binding affinity EC50 value measured according toExample 9 was 1194 ng/mL. The pharmacokinetic profile of the insulin-Fcfusion protein of SEQ ID NO: 36 was measured by the method of Example 12using ELISA, and a two-compartment model was fit to the data todetermine its elimination half-life which was 4.1±0.7 days. The sequenceof SEQ ID NO: 36 is shown below (B16A and cNg-NB151-S mutationsunderlined for clarity).

(SEQ ID NO: 36) FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHS PG

The insulin-Fc fusion protein of SEQ ID NO: 36 was then evaluated forrepeated dose bioactivity performance in dogs. The compound wasadministered subcutaneously to N=1 dog on day 0, day 7, day 14, and onday 28 according to the procedure of Example 11. When the dog's % FBGLdropped too low, the dog was given food to raise the blood glucose to asafe level. Unexpectedly, compared to the insulin-Fc fusion protein ofSEQ ID NO: 104, the NAOC for the first injection of the insulin-Fcfusion protein of SEQ ID NO: 36 containing the B16A mutation, wassignificantly higher (1185% FBGL·days·kg/mg). The first dose in vivobioactivity plot is shown in FIG. 26. The pharmacokinetic profile of thecompound was also measured by the method of Example 12 using ELISA, anda two-compartment model was fit to the data to determine its eliminationhalf-life which was 3.5 days. The NAOC and NAOCR were also measured foreach subsequent dose according to the general procedure of Example 11,calculated from the time the dose was administered until just before thenext dose was administered. The NAOC and the NAOCR shown in Table 18illustrate that the insulin-Fc fusion protein of SEQ ID NO: 36 maintainsan NAOCR greater than or equal to 0.6 throughout the four doses thusmeeting the repeated dose bioactivity design goal. Taken together, theresults indicate that it was necessary to mutate the insulin B-chainsequence to obtain a suitable, non-glycosylated cNg-S variant of SEQ IDNO: 32. Therefore, the insulin polypeptide of SEQ ID NO: 11 waspreferred for non-glycosylated insulin-Fc fusion proteins comprisingcNg-mutated canine IgGB Fc fragments.

TABLE 18 NAOC per dose for repeated doses of SEQ ID NO: 36 NAOCInjection# Day (% FBGL · days · kg/mg) NAOCR 1 0 1185 1.0 2 7 954 0.8 314 764 0.6 4 28 991 0.8

Finally, select compounds were tested for their likelihood to interactwith the immune system by measuring their Fc(gamma) receptor bindingactivity according to the procedure of Example 8. Table 19 compares theFc(gamma) receptor I binding of these insulin-Fc fusion proteins withthe Fc(gamma) receptor binding of the insulin-Fc fusion protein of SEQID NO: 52. It can be seen that the non-glycosylated insulin-Fc fusionproteins (achieved through a cNg-S mutation) exhibited the lowestFc(gamma) receptor binding ratio to SEQ ID NO: 52.

TABLE 19 Fc(gamma) receptor binding for cNg variations of SEQ ID NO: 52OD450 nm at a Fc(gamma)RI concentration OD450 nm Ratio to Species/FcGlycosylation of 3000 Minus Assay SEQ ID SEQ ID NO: Isotype Mutation(ng/mL) Background NO: 52 SEQ ID NO: 52 Canine/IgGB Native cNg 0.4280.371 1.00 SEQ ID NO: 32 Canine/IgGB Native cNg 0.368 0.311 0.84 SEQ IDNO: 96 Canine/IgGA Native cNg 0.253 0.196 0.53 SEQ ID NO: 104Canine/IgGB cNg-S 0.175 0.118 0.32 SEQ ID NO: 102 Canine/IgGB cNg-S and0.166 0.109 0.29 NB119-A SEQ ID NO: 36 Canine/IgGB cNg-S and 0.177 0.1200.32 B16A

Example 34: Exemplary CHO-Based Production Runs Using PreferredInsulin-Fc Fusion Proteins Comprising Fc Fragments of Canine IgGB OriginMade Via Stably Transfected CHO Cell Lines

Separate CHO cell lines stably transfected with vectors encoding for SEQID NO: 32, or SEQ ID NO: 36 were constructed as described in Example 2.Fed-batch shake flask 14-day production runs (0.5-2.0 L media scale)were seeded at 0.5 million cells/mL in an incubator-shaker set at 37° C.and 5% carbon dioxide, and the runs were conducted as described inExample 2 above, except that CD OptiCHO was substituted for Dynamis asthe growth media (ThermoFisher) and Efficient Feed C (ThermoFisher) wasused as the feed. Feed was added at 3% v/v starting on production runday 3, and on day 4, the shake-flask temperature was adjusted to 32° C.and the incubator-shaker carbon dioxide concentration was lowered from5% to 2%. During the run, the cells increased to between 8-14 millioncells/mL, and on Day 14 the production run was harvested to remove thecells and the culture supernatant was purified and tested to obtain theinsulin-Fc fusion protein as described in Examples 3, 4, 5, and 6. Table20 describes the manufacturing data obtained from the production runswith stably transfected CHO cell lines.

TABLE 20 Homodimer titers for non-glycosylated insulin-Fc fusionproteins of SEQ ID NO: 32 and SEQ ID NO: 36 Protein Yield HomodimerTiter SEQ ID NO: (mg/L) % Homodimer (mg/L) SEQ ID NO: 32 485 99.3% 482SEQ ID NO: 36 260 99.0% 257

Example 35: Exemplary CHO-Based Production Runs Using PreferredInsulin-Fc Fusion Proteins Comprising Fc Fragments of Canine IgGB OriginMade Via Stably Transfected CHO Cell Lines

A CHO cell line stably transfected with vectors encoding for SEQ ID NO:34 is constructed as described in Example 2. Fed-batch shake flask14-day production runs (0.5-2.0 L media scale) is seeded at 0 5 millioncells/mL in an incubator-shaker set at 37° C. and 5% carbon dioxide, andthe run is conducted as described in Example 2, except that CD OptiCHOis substituted for Dynamis as the growth media (ThermoFisher) andEfficient Feed C (ThermoFisher) is used as the feed. Feed is added at 3%v/v starting on production run day 3, and on day 4, the shake-flasktemperature is adjusted to 32° C. and the incubator-shaker carbondioxide concentration is lowered from 5% to 2%. On Day 14, theproduction run is harvested to remove the cells, and the culturesupernatant is purified and tested to obtain the insulin-Fc fusionprotein as described in Example 3, 4, 5, and 6. The resulting productionrun gives a protein yield of greater than 200 mg/L, greater than 95%homodimer, and greater than 190 mg/L homodimer titer of SEQ ID NO: 34.

Results—Insulin-Fc Fusion Proteins Comprising a Feline Fc FragmentExample 36: An Insulin-Fc Fusion Protein Comprising an Fc Fragment ofthe Feline IgG2 Isotype

To develop a product suitable for use in cats, an attempt was made toproduce an insulin-Fc fusion protein comprising the insulin polypeptidesequence of SEQ ID NO: 4 and the Fc fragment of the feline IgG2 isotype(SEQ ID NO: 21) using the peptide linker of SEQ ID NO: 13 with thefollowing amino acid sequence:

(SEQ ID NO: 106) FVNQHLCGSDLVEALYLVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGSGGGGGEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQTYWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG

The insulin-Fc fusion protein of SEQ ID NO: 106 was synthesized in HEKcells according to Example 1 and purified according to Example 3. Thestructure of the insulin-Fc fusion protein was confirmed according toExample 4 by non-reducing and reducing CE-SDS, and the sequence wasfurther identified by LC-MS with glycan removal according to Example 5.The % homodimer of the resulting compound, measured by size-exclusionchromatography according to Example 6, was 88%. The resulting homodimertiter was only 20 mg/L, which resulted from the inability for the HEKcells to make the product in high yield (i.e., the protein yield afterProtein a purification was only 23 mg/L). In summary, manufacturing ofthe insulin-Fc fusion protein of SEQ ID NO: 106 in HEK cells resulted ina moderate level of aggregates and a low homodimer titer of 20 mg/L,which did not meet the design goal of a homodimer titer of greater than50 mg/L.

Nevertheless, the insulin-Fc fusion protein of SEQ ID NO: 106 wasevaluated for bioactivity. First, the insulin receptor binding of theinsulin-Fc fusion protein of SEQ ID NO: 106 was measured according toExample 7, resulting in an IC50 value of 22 nM indicating that thecompound is likely to be bioactive in vivo (i.e., IC50 less than 5000nM).

Next, the in vivo pharmacodynamics (PD) of the insulin-Fc fusion proteinof SEQ ID NO: 106 was measured after a single subcutaneousadministration of the compound to N=3 cats at a dose of 0.8 mg/kgaccording to Example 10. FIG. 27 shows the percent fasting blood glucoselevel for the insulin-Fc fusion protein of SEQ NO: 106 (161c) as afunction of time. The NAOC for the insulin-Fc fusion protein wascalculated to be 215% FBGL·days·kg/mg according to the procedure ofExample 11. Surprisingly, unlike the analogous insulin-Fc fusion proteinfor dogs of SEQ ID NO: 42 comprising the insulin polypeptide of SEQ IDNO: 5 and the peptide linker of SEQ ID NO: 12, the insulin-Fc fusionprotein for cats of SEQ NO: 106 was found to be much less aggregated andsignificantly more bioactive in the target animal.

Since the NAOC was acceptable and the pharmacokinetic data wassupportive of a once-weekly administration, the cats were givenadditional subcutaneous doses on day 28, day 35, day 42 and day 49 andthe % FBGL was measured for the 7-day window after each dose accordingto Example 11. The NAOC and NAOCR were calculated according to theprocedure of Example 11 for each repeated subcutaneous injection. Asillustrated in Table 21, repeated subcutaneous dosing in cats revealed asignificant decay in bioactivity by the third dose as measured by asignificant decrease in the NAOCR (i.e., the NAOC for the thirdinjection was only 0.40, or 40%, of the NAOC for the first injection,and the NAOC for the fourth injection was only 0.10, or 10%, of the NAOCfor the first injection). The significant decay in bioactivity for theinsulin-Fc fusion protein of SEQ ID NO: 106 after repeated dosing incats was similar to that observed for the insulin-Fc fusion protein ofSEQ ID NO: 52 in dogs shown in Example 20.

TABLE 21 NAOC per dose for repeated doses of SEQ ID NO: 106 NAOCInjection# Day (% FBGL · days · kg/mg) NAOCR 1 0 215 1.0 2 28 161 0.7 335 120 0.6 4 42 80 0.4 5 49 21 0.1

Example 37: Evaluation of Insulin Polypeptide Mutations and the Choiceof Feline IgG1b or IgG2 Fc Fragments on Protein Yield, Purity, andInsulin Receptor Activity

In an attempt to increase the % homodimer content and protein yield ofthe insulin-Fc fusion protein of SEQ ID NO: 106, mutations were insertedinto the sequences of the insulin polypeptide B-chain (e.g., the B16Amutation) and the peptide linker. Furthermore, the feline IgG1b Fcfragment (SEQ ID NO: 20) was evaluated in addition to the feline IgG2 Fcfragment (SEQ ID NO: 21) that was used to construct the insulin-Fcfusion protein of SEQ ID NO: 106. The resulting insulin-Fc fusionprotein sequences are shown below with the resulting sequence alignmentsagainst SEQ ID NO: 106 shown in FIG. 28 (Clustal Omega).

(SEQ ID NO: 108) FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGSGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 110)FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGAGGGGGEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 112)FVNQHLCGSDLVEALALVCGERGFFYTDPTGGGPRRGIVEQCCHSICSLYQLENYCNGGGGSGGGGGEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG

The insulin-Fc fusion proteins were manufactured in HEK293 cellsaccording to Example 1 and purified using a Protein A column accordingto Example 3. Their structures were confirmed according to Example 4 bynon-reducing and reducing CE-SDS, and the sequences were furtheridentified by LC-MS with glycan removal according to Example 5. Their %homodimer content was measured by size-exclusion chromatographyaccording to Example 6, and their insulin receptor binding affinitieswere measured according to Example 7. The insulin-Fc fusion proteinvariants are listed in Table 22 along with the corresponding proteinyields, % homodimer, and homodimer titer. The results show that thevarious mutations, when combined with the feline IgG1b isotype Fcfragment to produce the insulin-Fc fusion protein of SEQ ID NO: 108,gave rise to a much higher protein yield, but the resulting protein wasmore aggregated (e.g. lower % homodimer than SEQ ID NO: 106). This wassurprising as the feline IgG1b is more similar in function to the canineIgGB Fc fragment isotype, which was the highly preferred Fc isotype forthe production of canine insulin-Fc fusion proteins (Example 32). Of themutated feline compositions containing the feline IgG2 isotype, the onescomprising B16A mutation of the insulin polypeptide B-chain (i.e., SEQID NO: 110 and SEQ ID NO: 112) led to improved protein yield andhomodimer titers. However, the mutated linker present in SEQ ID NO: 110(i.e., GGGGAGGGG (SEQ ID NO: 12)) seems to have provided a furtherdoubling in protein yield and homodimer titer as compared to SEQ ID NO:112.

TABLE 22 Manufacturing and IR Binding for insulin-Fc fusion proteinsutilizing feline IgG1b and IgG2 Fc fragments Homo- IR % dimer Binding,SEQ ID IgG Protein Homo- Titer IC50 NO: Fragment (mg/L) dimer (mg/L)(nM) SEQ ID IgG2 23 88.0% 20 22 NO: 106 SEQ ID IgG1b 127 49.0% 62 62 NO:108 SEQ ID IgG2 122 89.7% 109 41 NO: 110 SEQ ID IgG2 64 80.4% 51 53 NO:112

Example 38: In Vivo Immunogenicity Screening after Repeated SubcutaneousDoses of the Insulin-Fc Fusion Protein Comprising the InsulinPolypeptide of SEQ ID NO: 4 with a Feline IgG2 Isotype Fc Fragment

Without being bound to any particular explanation, it was postulatedthat the cause of the significant reduction in bioactivity of theinsulin-Fc fusion protein of SEQ ID NO: 106 after the fourth repeatedsubcutaneous dose in cats (Example 36) was due to the development ofanti-drug antibodies that neutralized its biological activity. Anti-drugantibodies may be directed against the insulin polypeptide, linker, orFc-fragment portions of an insulin-Fc fusion protein. The immunogenicresponse manifests as interactions between antigen presenting cells,T-helper cells, B-cells, and their associated cytokines, which may leadto the production of endogenous antibodies against the drug (e.g.anti-drug antibodies). Binding antibodies are all isotypes capable ofbinding the insulin-Fc fusion protein, and these may be detected in animmunoassay as described in Example 14. Neutralizing antibodies thatinhibit functional activity of the insulin-Fc fusion protein aregenerally directed against a biologically active site. To assess whetherthis was the case, serum that was collected prior to the administrationof each dose and at the end of the experiment described in Example 11was tested to quantify the levels of anti-drug antibodies according toExample 14. As shown in FIG. 29, levels of anti-drug antibodies didindeed increase with multiple subcutaneous administrations of thecompound, indicating that the generation of neutralizing anti-drugantibodies was the likely cause for the reduction in the NAOCR after thefourth injection of the insulin Fc-fusion protein of SEQ ID NO: 106.

Example 39: Screening of Feline Serum Containing Anti-Drug Antibodiesand Identification of Potential Immunogenic Epitopes at the B10D and A8HPositions of the Insulin Polypeptide

As was observed for SEQ ID NO: 52 in dogs (Example 20), the repeateddose bioactivity of the insulin-fusion protein of SEQ ID NO: 106comprising the insulin polypeptide of SEQ ID NO: 4 and the peptidelinker of SEQ ID NO: 13 still gave rise to anti-drug antibodies (Example38). It was hypothesized, therefore, that the insulin polypeptide of SEQID NO: 4 may unexpectedly contain specific epitopes (i.e., immunogenic“hot spots”) against which a cat's immune system is directed. Therefore,the binding specificity of the antibodies present in the serum samplesdescribed in Example 38 were evaluated according to the generalprocedure of Example 15. The analysis of the antibody-containing felineserum samples from the repeated dosing of the insulin-Fc fusion proteinof SEQ ID NO: 106 (Example 38) against the coated insulin-Fc fusionprotein library demonstrated that there were unexpectedly two primary“hot spots” present within the insulin polypeptide sequence of SEQ IDNO: 4: the B10D site mutation (i.e., the aspartic acid mutation at the10th position from the N-terminus of the B-chain (i.e., B10)), and,separately, the A8H site mutation (i.e., the histidine mutation at the8th position from the N-terminal end of the A-chain (i.e., A8)). Theresults suggest that insulin-Fc fusion proteins comprising insulinpolypeptide amino acid compositions containing these two particularamino acid mutations are likely to be immunogenic in cats and thereforelikely to give rise anti-drug antibodies that neutralize the bioactivityafter repeated injections. Therefore, it was determined that insulinpolypeptides that do not contain the B10D and A8H are preferred forinsulin-Fc fusion proteins that need to be repeatedly dosed in cats overlong periods long-term (e.g., to treat feline diabetes).

Example 40: Insulin-Fc Fusion Proteins Comprising the InsulinPolypeptide of SEQ ID NO: 4 and Glycosylated and Non-Glycosylated FelineIgG1b and IgG2 Isotype Fc Fragments in which the B10, A8, and OtherSites of the Insulin Polypeptide are Further Mutated to Reduce thePotential Risk of Immunogenicity

To evaluate whether replacing the “hot spot” mutations would improve theimmunogenicity and repeated dose bioactivity of insulin-Fc fusionproteins comprising the insulin polypeptide of SEQ ID NO: 4 and thefeline IgG2 isotype fragment, exemplary insulin-Fc fusion proteins ofSEQ ID NOs: 114, 116, and 118 were synthesized in which the B10 and A8amino acids of the insulin polypeptide were restored to their nativehistidine and alanine compositions, respectively, and the histidine atB16 was replaced with alanine (i.e., B16A) as was the case for theinsulin polypeptide of SEQ ID NO: 5 used for many of the canineinsulin-Fc fusion proteins. The A21N site of the native insulin was alsodeleted. For this example, other insulin polypeptide amino acids weremutated to make the structure more similar to native feline insulin(e.g., B30A, ABA, A10V, and A18H). The sequence of the resulting insulinpolypeptide (SEQ ID NO: 120) is listed below with the non-native aminoacids to feline insulin underlined.

(SEQ ID NO: 120) FVNQHLCGSHLVEALALVCGERGFFYTDPAGGGPRRGIVEQCCASVCSLYQLEHYCFurthermore, given the additional potential benefits of thenon-glycosylated cNg mutants discussed in Examples 22 and 33, two of theevaluated insulin-Fc fusion proteins (SEQ ID NOs: 116 and 118) containthe cNg-S mutation. The entire amino acid sequences of the insulin-Fcfusion proteins are shown below with the resulting sequence alignmentsagainst SEQ ID NO: 108 shown in FIG. 30 (Clustal Omega).

(SEQ ID NO: 114) FVNQHLCGSHLVEALALVCGERGFFYTDPAGGGPRRGIVEQCCASVCSLYQLEHYCGGGGAGGGGGEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSP (SEQ ID NO: 116)FVNQHLCGSHLVEALALVCGERGFFYTDPAGGGPRRGIVEQCCASVCSLYQLEHYCGGGGAGGGGGEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFSSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPG (SEQ ID NO: 118)FVNQHLCGSHLVEALALVCGERGFFYTDPAGGGPRRGIVEQCCASVCSLYQLEHYCGGGGAGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVALGPDDSDVQITWFVDNTQVYTAKTSPREEQFSSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG

The insulin-Fc fusion proteins were manufactured in HEK293 cellsaccording to Example 1 and purified using a Protein A column accordingto Example 3. Their structures were confirmed according to Example 4 bynon-reducing and reducing CE-SDS, and the sequences were furtheridentified by LC-MS with glycan removal according to Example 5. Their %homodimer content was measured by size-exclusion chromatographyaccording to Example 6, and their insulin receptor binding affinitieswere measured according to Example 7. Table 23 below illustrates themanufacturability and in vitro IR binding parameters for the resultingcompounds.

TABLE 23 Manufacturing and IR Binding for insulin-Fc fusion proteinsutilizing feline IgG1b and IgG2 Fc fragments Protein Homodimer IRBinding, IgG Yield Titer IC50 SEQ ID NO: Fragment (mg/L) % Homodimer(mg/L) (nM) SEQ ID NO: 108 IgG1b 127 48.6% 62 62 SEQ ID NO: 118 IgG1b 1897.5% 18 >5000 SEQ ID NO: 114 IgG2 25 90.5% 23 3,480 SEQ ID NO: 116 IgG21 73.0% 1 707

Unexpectedly, all three insulin-Fc fusion proteins gave much lowerprotein yields compared to that of the insulin-Fc fusion protein of SEQID NO: 108. In fact, although it had a sufficiently high insulinreceptor binding affinity (IC50 of 707 nM), the insulin-Fc fusionprotein of SEQ ID NO: 116 gave almost no protein yield. The insulin-Fcfusion protein of SEQ ID NO: 118 gave unacceptably low protein yield andhomodimer titer and was deemed unlikely to be bioactive in vivo due toits high IR binding IC50 value greater than 5000 nM. The protein of SEQID NO: 114 also gave an unacceptably low protein yield and a much lowerinsulin receptor binding affinity (higher IR IC50 value) compared tothat of the insulin-Fc fusion protein of SEQ ID NO: 108.

Example 41: An Insulin-Fc Fusion Protein Comprising the InsulinPolypeptide of SEQ ID NO: 8, Linker of SEQ ID NO: 14 and a Feline IgG2Isotype Fc Fragment

In an attempt to obtain an acceptable protein yield of an insulin-Fcfusion protein comprising an insulin polypeptide sequence without theimmunogenic “hot spot” mutations (i.e., B10D and A8H), learnings wereobtained from the simultaneous and parallel development of canineinsulin-Fc fusion proteins that had shown that the use of an insulinpolypeptide of SEQ ID NO: 8 and a peptide linker of SEQ ID NO: 14 on acanine IgGB isotype Fc fragment resulted in high protein and homodimertiters and acceptable IR binding affinity. Therefore, a felineinsulin-Fc fusion protein was constructed using the insulin polypeptideof SEQ ID NO: 8 and the peptide linker of SEQ ID NO: 14 on a feline IgG2Fc fragment of SEQ ID NO: 21 to produce the following sequence:

(SEQ ID NO: 122) FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGGEGPKCPVPEIPGAPSVFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSNVQITWFVDNTEMHTAKTRPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSAMERTISKAKGQPHEPQVYVLPPTQEELSENKVSVTCLIKGFHPPDIAVEWEITGQPEPENNYQTTPPQLDSDGTYFLYSRLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQ SPGThe sequence alignment of SEQ ID NO: 122 against the Example 37sequences SEQ ID NOs: 106 and 112 are shown in FIG. 31 (Clustal Omega).

TABLE 24 Manufacturing and IR Binding for insulin-Fc fusion proteinsutilizing feline IgG1b and IgG2 Fc fragments Protein Homodimer IRBinding, IgG Yield Titer IC50 SEQ ID NO: Fragment (mg/L) % Homodimer(mg/L) (nM) SEQ ID NO: 106 IgG2 23 88.0% 20 22 SEQ ID NO: 112 IgG2 6480.4% 51 53 SEQ ID NO: 122 IgG2 146 99.0% 145 2,536

The insulin-Fc fusion protein of SEQ ID NO: 122 was manufactured inHEK293 cells according to Example 1 and purified using a Protein Acolumn according to Example 3. Their structures were confirmed accordingto Example 4 by non-reducing and reducing CE-SDS, and the sequences werefurther identified by LC-MS with glycan removal according to Example 5.Their % homodimer content was measured by size-exclusion chromatographyaccording to Example 6, and their insulin receptor binding affinitieswere measured according to Example 7. The FcRn receptor binding affinitywas measured according to Example 9. The protein yield was 146 mg/L, andthe % homodimer was determined to be 99%, resulting in a homodimer titerof 145 mg/L which meets the manufacturing design goal. The IR bindingaffinity IC50 value was 2,536 nM indicating that the compound is likelyto be bioactive in vivo. The FcRn receptor binding affinity EC50 valuewas 3114 ng/mL. Therefore, the insulin-Fc fusion protein of SEQ ID NO:122 was a potential candidate for further testing in vivo.

Example 42: In Vivo Bioactivity of an Insulin-Fc Fusion ProteinConstructed from the Insulin Polypeptide of SEQ ID NO: 8, the PeptideLinker of SEQ ID NO: 14, and the Feline IgG2 Fc Fragment of SEQ ID NO:21

The insulin-Fc fusion protein of SEQ ID NO: 122 was tested forbioactivity in vivo according to Example 10. A healthy, antibody-naïve,cat weighing approximately 5 kg was used. On day 0 the cat received asingle injection of a pharmaceutical composition containing the insulinFc-fusion protein of SEQ ID NO: 122. On day 0, blood was collected froma suitable vein immediately prior to injection and at 15, 30, 45, 60,120, 240, 360, and 480 min and at 1, 2, 3, 4, 5, 6, and 7 days postinjection. If the subject's blood glucose dropped to dangerous levels,food and/or dextrose injections were given to prevent symptomatichypoglycemia.

FIG. 32 shows the % FBGL for a single administration, illustrating that,unexpectedly, the insulin-Fc fusion protein of SEQ ID NO: 122 was onlymarginally bioactive in vivo (NAOC of essentially 0% FBGL·days·kg/mg).This result was surprising, especially since the insulin-Fc fusionprotein was not aggregated (i.e., had a high % homodimer content), andthe molecule exhibited an IR affinity in a similar range as the canineinsulin-Fc fusion proteins that were found to exhibit significantbioactivity in dogs (Example 31). Due to the lack of bioactivity on thefirst administration, repeat administrations were not performed.

Example 43: Evaluation of the Substitution of Feline IgG1b for theFeline IgG2 Fc Fragment on the Yield, Purity, Bioactivity, andImmunogenicity of an Insulin-Fc Fusion Protein Comprising the InsulinPolypeptide of SEQ ID NO: 8 and the Peptide Linker of SEQ ID NO: 14

Because the dog and cat long-acting insulin research programs wereconducted in parallel, some of the learnings of the canine insulin-Fcfusion protein research program were applied to the feline insulin-Fcprotein research program. One key learning from the canine insulin-Fcresearch program was how the selection of different IgG isotype Fcfragments (e.g. canine IgGA, canine IgGB, canine IgGC, and canine IgGDisotypes) led to dramatically different manufacturing and in vivoefficacy performance Therefore, the feline IgG2 Fc fragment of SEQ IDNO: 122 was replaced with the feline IgG1b Fc fragment of SEQ ID NO: 20while keeping the insulin polypeptide of SEQ ID NO: 8 and the peptidelinker of SEQ ID NO: 14 resulting in the following amino acid sequence:

(SEQ ID NO: 38) FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQS PG

The insulin-Fc fusion protein of SEQ ID NO: 38 was synthesized in HEK293cells according to the procedure of Example 1 and purified using aProtein A column according to Example 3. The structure was confirmedaccording to Example 4 by non-reducing and reducing LC-MS, and thesequence was further identified by LC-MS with glycan removal accordingto Example 5. The protein yield was 158 mg/L at this stage. The %homodimer for the sequence was measured by size-exclusion chromatographyaccording to Example 6 and was determined to be 99.5% resulting in ahomodimer titer of 157 mg/L which meets the manufacturing design goal.The in vitro IM-9 insulin receptor binding IC50 value, measuredaccording to Example 7, was 2398 nM which also meets the design goal.The FcRn receptor binding affinity EC50 value was measured according toExample 9 and found to be 1552 ng/mL.

The insulin-Fc fusion protein of SEQ ID NO: 38 was then tested forbioactivity in vivo according to Example 10. A healthy, antibody-naïve,cat weighing approximately 5 kg received a single subcutaneous injectionof a pharmaceutical composition containing the insulin Fc-fusion proteinof SEQ ID NO: 38 at a dose of 0.16 mg insulin-Fc fusion protein/kg. Onday 0, blood was collected from a suitable vein immediately prior toinjection and at 15, 30, 45, 60, 120, 240, 360, and 480 min and at 1, 2,3, 4, 5, 6, and 7 days post injection. If the subject's blood glucosedropped to dangerous levels, food and/or dextrose injections were givento prevent symptomatic hypoglycemia.

FIG. 33 shows the % FBGL after the first administration. Food was givento the animal regularly to prevent symptomatic hypoglycemia,illustrating that the insulin-Fc fusion protein of SEQ ID NO: 38 wassignificantly bioactive in vivo with a NAOC of 1838% FBGL·days·kg/mg.The pharmacokinetic profile of the compound was also measured by themethod of Example 12 using ELISA, and a two-compartment model was fit tothe data to determine its elimination half-life which was 6.3±0.5. Thedifference in biological activity (in vitro and in vivo) between theinsulin-Fc fusion protein of SEQ ID NO: 38 and that of SEQ ID NO: 122demonstrates that, unexpectedly, the feline IgG1b isotype is preferredover the feline IgG2 isotype for the Fc fragment when the insulinpolypeptide sequence is modified as in SEQ ID NO: 8.

Since the NAOC was acceptable and the pharmacokinetic data wassupportive of a once-weekly administration, the cat was given additionalsubcutaneous doses on day 14, day 28, and on day 42, and the % FBGL wasmeasured for the 7-day window after each dose according to Example 11.The NAOC and NAOCR were calculated according to the procedure of Example11 for each repeated subcutaneous injection. As illustrated in Table 25,the insulin-Fc fusion protein of SEQ ID NO: 38 demonstrated acceptablebioactivity in vivo after multiple doses.

TABLE 25 NAOC per dose for repeated doses of SEQ ID NO: 38 NAOCInjection# Day (% FBGL · days · kg/mg) NAOCR 1 0 1838 1.0 2 14 1431 0.83 28 1900 1.0 4 42 2400 1.3

In addition, serum was collected prior to the administration of eachdose and once a week for two weeks after the end of the experiment inorder to test for the presence and quantify the levels of any anti-drugantibodies according to Example 14. As shown in FIG. 34, there was nomeasurable increase in anti-drug antibodies above baseline aftermultiple administrations of the compound. Therefore, in order to obtaina feline insulin-Fc fusion protein candidate (e.g. SEQ ID NO: 38) thatmeets the design criteria of acceptable homodimer titer, in vivobioactivity, and sustained bioactivity after repeated weekly injectionsin cats, it was necessary to replace the insulin polypeptide of SEQ IDNO: 4 with the insulin polypeptide of SEQ ID NO: 8 and use the felineIgG1b Fc fragment of SEQ ID NO: 20 instead of the feline IgG2 Fcfragment of SEQ ID NO: 21.

Example 44: Non-Glycosylated Insulin-Fc Fusion Proteins Comprising theInsulin Polypeptide of SEQ ID NO: 8, the Peptide Linker of SEQ ID NO:14, and the Feline IgG1b Fc Fragment to Reduce the Potential Risk ofImmunogenicity

While the insulin-Fc fusion protein of SEQ ID NO: 38 meets all of thedesign goals (Example 43), there may or may not be a risk ofimmunogenicity over extended periods of treatment (e.g., 6 months, 1year, 2 years or more), which could compromise the use of thisinsulin-Fc fusion protein for treating diabetes should this occur. Asdescribed in the Detailed Description of the Invention, one possiblecause of a reduction in bioactivity after repeated doses is the unwantedinteraction of the feline IgG1b Fc fragment with the cat's immune systemresulting in the production of neutralizing anti-drug antibodies.However, the results shown in Example 43 demonstrate that unexpectedly,the feline IgG1b isotype was preferable over the less immunogenic felineIgG2 isotype with respect to in vivo bioactivity. Therefore, further Fcmutations were explored to achieve non-glycosylated insulin-Fc fusionproteins with low Fc(gamma)RI receptor binding, which should reduce thelong-term, chronic immunogenicity risk.

As described in the Detailed Description of the Invention, one methodfor reducing the Fc(gamma)RI interaction involves mutating the Fcfragment cNg site to prevent glycosylation during synthesis in the hostcell. Therefore, cNg site mutations were made to the Fc fragment regionof SEQ ID NO: 38 to reduce the binding affinity of the Fc fragment forFc(gamma) receptors in vivo, as measured by binding in an in vitro humanFc(gamma)RI assay described in Example 8. The position of the cNg sitein the insulin-Fc fusion protein of SEQ ID NO: 38 is cNg-NB151. Again,capitalizing on the learnings from the canine insulin-Fc fusion proteinsdescribed in Example 33, a cNg-NB151-S mutation was introduced into theFc fragment of SEQ ID NO: 38. The full amino acid sequence of theresulting insulin-Fc fusion protein is listed below (cNg-NB151-Sunderlined for clarity):

(SEQ ID NO: 124) FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFSSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQS PG

The insulin-Fc fusion protein of SEQ ID NO: 124 was synthesized inHEK293 cells according to the procedure of Example 1 and purified usinga Protein A column according to Example 3. The structure of theinsulin-Fc fusion protein was confirmed according to Example 4 bynon-reducing and reducing LC-MS, and the sequence was further identifiedby LC-MS with glycan removal according to Example 5. The protein yieldwas 202 mg/L at this stage. The % homodimer for the sequence wasmeasured by size-exclusion chromatography according to Example 6 and wasdetermined to be 99%, resulting in a homodimer titer of 200 mg/L whichmeets the manufacturing design goal. However, the in vitro IM-9 insulinreceptor binding IC50 value, measured according to Example 7, wasgreater than 5000 nM, which is outside the design goal for in vitrobioactivity. The FcRn receptor binding affinity EC50 value was measuredaccording to Example 9 and was 6922 ng/mL.

Although the insulin-Fc fusion protein of SEQ ID NO: 124 did not meetthe insulin receptor binding design goal, it was tested for bioactivityin vivo according to Example 10. A healthy, antibody-naïve, cat weighingapproximately 5 kg was used. On day 0 the cat received a singleinjection of a pharmaceutical composition containing the insulinFc-fusion protein of SEQ ID NO: 124 at a dose of 0.16 mg insulin-Fcfusion protein/kg. On day 0, blood was collected from a suitable veinimmediately prior to injection and at 15, 30, 45, 60, 120, 240, 360, and480 min and at 1, 2, 3, 4, 5, 6, and 7 days post injection. If thesubject's blood glucose dropped to dangerous levels, food and/ordextrose injections were given to prevent symptomatic hypoglycemia.

FIG. 35 shows the % FBGL for a single administration, illustrating thatthe insulin-Fc fusion protein of SEQ ID NO: 124 is only somewhatbioactive in vivo with an NAOC of 65% FBGL·days·kg/mg. Due to the lackof bioactivity on the first administration, repeat administrations werenot performed.

Unexpectedly, as was the case in Example 33 for the canine insulinFc-fusion protein of SEQ ID NO: 36, it was found that mutating theinsulin polypeptide sequence of SEQ ID NO: 124 such that the 16th aminoacid from the N-terminus of the B-chain (B16) was mutated from tyrosineto alanine (i.e., B16A) rendered the resulting insulin-Fc fusion proteinof SEQ ID NO: 40 bioactive. The amino acid sequence of the resultinginsulin-Fc fusion protein is shown below (B16A and cNg-NB151-S mutationsunderlined for clarity):

(SEQ ID NO: 40) FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFSSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQS PG

The insulin-Fc fusion protein of SEQ ID NO: 40 was synthesized in HEK293cells according to the procedure of Example 1 and purified using aProtein A column according to Example 3. The structure of the insulin-Fcfusion protein was confirmed according to Example 4 by non-reducing andreducing CE-SDS, and the sequence was further identified by LC-MS withglycan removal according to Example 5. The protein yield was 174 mg/L atthis stage. The % homodimer for the sequence was measured bysize-exclusion chromatography according to Example 6 and was determinedto be 98.9% resulting in a homodimer titer of 172 mg/L which meets themanufacturing design criteria. The in vitro IM-9 insulin receptorbinding IC50 value of 4635 nM, measured according to Example 7, alsomeets the design goal. The Fc(gamma) receptor activity was measuredaccording to Example 8 and found to be approximately four times lessthan that obtained for the insulin-Fc fusion protein of SEQ ID NO: 38using the same procedure indicating that the insulin-Fc fusion proteinis less likely to adversely interact with the cat's immune system. TheFcRn receptor binding affinity EC50 value was measured according toExample 9 and was 8157 ng/mL.

The insulin-Fc fusion protein of SEQ ID NO: 40 was then tested forbioactivity in vivo according to Example 11. A healthy, antibody-naïve,cat weighing approximately 5 kg was used. On day 0, day 7, and day 21the cat received a single subcutaneous injection of a pharmaceuticalcomposition containing an insulin Fc-fusion protein of SEQ ID NO: 40 ata dose of 0.1 mg insulin-Fc fusion protein/kg. On day 0, blood wascollected from a suitable vein immediately prior to injection and at 15,30, 45, 60, 120, 240, 360, and 480 min and at 1, 2, 3, 4, 5, 6, and 7days post injection. If the subject's blood glucose dropped to dangerouslevels, food and/or dextrose injections were given to preventsymptomatic hypoglycemia.

FIG. 36 shows the % FBGL after the first administration, illustratingthat the insulin-Fc fusion protein of SEQ ID NO: 40 is bioactive in vivowith a NAOC of 159% FBGL·days·kg/mg for a subcutaneous dose of 0.1 mginsulin-Fc fusion protein/kg. A second higher subcutaneous dose of 0.2mg insulin-Fc fusion protein/kg gave a much higher NAOC of 702%FBGL·days·kg/mg and is shown in FIG. 37. The pharmacokinetic profile ismeasured by the method of Example 12 using ELISA, and a two-compartmentmodel is fit to the data to determine its elimination half-life which isgreater than 3 days. These results are in contrast to the resultsobtained with the insulin-Fc fusion protein of SEQ ID NO: 124 whichshowed that the same compound comprising a tyrosine at B16 instead of analanine was only very weakly bioactive at approximately the same dose(0.16 mg insulin-Fc fusion protein/kg). Therefore, the insulinpolypeptide of SEQ ID NO: 11 was preferred for non-glycosylatedinsulin-Fc fusion proteins comprising cNg mutated feline IgG1b Fcfragments.

To analyze the repeatable bioactivity after multiple doses, the cat wasgiven a further dose of the insulin-Fc fusion protein of SEQ ID NO: 40on day 7, on day 21, and on day 35. When the cat's % FBGL dropped toolow, the cat was given food to raise the blood glucose to a safe level.The NAOC and NAOCR were measured for each subsequent dose according tothe general procedure of Example 11, calculated from the time the dosewas administered until just before the next dose was administered. TheNAOC and the NAOCR shown in Table 26 illustrate that the insulin-Fcfusion protein of SEQ ID NO: 40 is bioactive in vivo after multipledoses.

TABLE 26 NAOC per dose for repeated doses of SEQ ID NO: 40 NAOCInjection# Day (% FBGL · days · kg/mg) NAOCR 1 0 159 1.0 2 7 702 4.4 321 462 2.9 4 35 670 4.2

In addition, serum was collected prior to the administration of eachdose and at the end of the experiment in order to test for the presenceand quantify the levels of any anti-drug antibodies according to Example14. There is no measurable increase in anti-drug antibodies abovebaseline after multiple administrations of the compound. Therefore, inorder to obtain a feline insulin-Fc fusion protein meeting themanufacturing and bioactivity design criteria with significantly reducedFc(gamma) receptor activity, it was not only necessary to mutate the cNgto serine but also to mutate the insulin polypeptide B16 amino acid toalanine.

Example 45: Exemplary CHO-Based Production Runs Using PreferredInsulin-Fc Fusion Proteins Comprising Fc Fragments of Feline IgG1bOrigin Made Via Stably Transfected CHO Cell Lines

A CHO cell line stably transfected with vectors encoding for SEQ ID NO:38 was constructed as described in Example 2 above. Fed-batch shakeflask 14-day production runs (0.5-2.0 L media scale) were seeded at 0.5million cells/mL in an incubator-shaker set at 37° C. and 5% carbondioxide, and the runs were conducted as described in Example 2 above,except that CD OptiCHO was substituted for Dynamis as the growth media(ThermoFisher) and Efficient Feed C (ThermoFisher) was used as the feed.Feed was added at 3% v/v starting on production run day 3, and on day 4,the shake-flask temperature was adjusted to 32° C. and theincubator-shaker carbon dioxide concentration was lowered from 5% to 2%.During the run, the cell density increased to between 8-14 millioncells/mL, and on Day 14 the production run was harvested to remove thecells, and the culture supernatant was purified and characterized toobtain the insulin-Fc fusion protein as described in Example 3, 4, 5,and 6. Table 27 describes the manufacturing data for the insulin-Fcfusion protein obtained via these stably transfected CHO cell lineproduction runs.

TABLE 27 Homodimer titers for non-glycosylated insulin-Fc fusionproteins of SEQ ID NO: 38 Protein Yield Homodimer Titer SEQ ID NO:(mg/L) % Homodimer (mg/L) SEQ ID NO: 38 633 96.3% 610

Example 46: Exemplary CHO-Based Production Runs Using PreferredInsulin-Fc Fusion Proteins of Feline IgG1b Origin Made Via StablyTransfected CHO Cell Lines

A CHO cell line stably transfected with vectors encoding for SEQ ID NO:40 is constructed as described in Example 2 above. A fed-batch shakeflask 14-day production run (0.5-2.0 L media scale) is seeded at 0.5million cells/mL in an incubator-shaker set at 37° C. and 5% carbondioxide, and the run is conducted as described in Example 2 above,except that CD OptiCHO is substituted for Dynamis as the growth media(ThermoFisher) and Efficient Feed C (ThermoFisher) is used as the feed.Feed is added at 3% v/v starting on production run day 3, and on day 4,the shake-flask temperature is adjusted to 32° C. and theincubator-shaker carbon dioxide concentration is lowered from 5% to 2%.On Day 14, the production run is harvested to remove the cells, and theculture supernatant is purified and characterized to obtain theinsulin-Fc fusion protein as described in Example 3, 4, 5, and 6. Theresulting production run gives a protein yield of greater than 200 mg/L,greater than 95% homodimer, and greater than 190 mg/L homodimer titer ofSEQ ID NO: 40.

Example 47: Exemplary Insulin-Fc Fusion Protein Domains and Sequences

Exemplary insulin-Fc fusion protein amino acid sequences andcorresponding DNA sequences used in the above Examples are shown FIGS.38, 39, 40, 41, and 42.

EQUIVALENTS

In the claims, articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The disclosure includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Thedisclosure includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

Furthermore, the disclosure encompasses all variations, combinations,and permutations in which one or more limitations, elements, clauses,and descriptive terms from one or more of the listed claims areintroduced into another claim. For example, any claim that is dependenton another claim can be modified to include one or more limitationsfound in any other claim that is dependent on the same base claim. Whereelements are presented as lists, e.g., in Markush group format, eachsubgroup of the elements is also disclosed, and any element(s) can beremoved from the group. It should be understood that, in general, wherethe disclosure, or aspects of the disclosure, is/are referred to ascomprising particular elements and/or features, certain embodiments ofthe disclosure or aspects of the disclosure consist, or consistessentially of, such elements and/or features. For purposes ofsimplicity, those embodiments have not been specifically set forth inhaec verba herein. It is also noted that the terms “comprise(s),”“comprising,” “contain(s),” and “containing” are intended to be open andthe use thereof permits the inclusion of additional elements or steps.Where ranges are given, endpoints are included. Furthermore, unlessotherwise indicated or otherwise evident from the context andunderstanding of one of ordinary skill in the art, values that areexpressed as ranges can assume any specific value or sub-range withinthe stated ranges in different embodiments of the disclosure, to thetenth of the unit of the lower limit of the range, unless the contextclearly dictates otherwise.

We claim:
 1. A fusion protein comprising an insulin polypeptide and anFc fragment, wherein the insulin polypeptide and the Fc fragment areconnected by a linker, wherein the Fc fragment comprises the followingsequence: (SEQ ID NO: 23)DCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFSSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQSPG

and wherein the insulin polypeptide comprises the following sequence:(SEQ ID NO: 10) FVNQHLCGSX₁LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCX₂STCSLDQLENYC

and wherein X₁ is not D and X₂ is not H.
 2. The fusion protein of claim1, wherein the insulin polypeptide comprises the following sequence:(SEQ ID NO: 10) FVNQHLCGSX₁LVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCX₂STCSLDQLENYC

wherein X₁ is H and X₂ is T.
 3. The fusion protein of claim 1 comprisingdomains in the following orientation from N- to C-terminus:(N-terminus)--insulin polypeptide--linker--Fc fragment--(C-terminus). 4.The fusion protein of claim 1, wherein the insulin polypeptide and theFc fragment are connected by a linker, comprising the followingsequence: (SEQ ID NO: 14) GGGGGQGGGGQGGGGQGGGGG.


5. A fusion protein comprising an insulin polypeptide linked to an Fcfragment, wherein the fusion protein comprises the following sequence:(SEQ ID NO: 40) FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFSSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKDKGQPHEPQVYVLPPAQEELSRNKVSVTCLIEGFYPSDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFLYSRLSVDRSRWQRGNTYTCSVSHEALHSHHTQKSLTQS PG.


6. The fusion protein of claim 1, wherein the fusion protein is ahomodimer.
 7. The fusion protein of claim 6, wherein the percentagehomodimer of the fusion protein is greater than 90%.
 8. The fusionprotein of claim 6, wherein the fusion protein is made using one ofHEK293 or CHO cells, and the resulting homodimer titer afterpurification using Protein A beads or a Protein A column is greater than50 mg/L.
 9. The fusion protein of claim 1, wherein the insulin receptorIC50 for the fusion protein is less than or equal to 5000 nM.
 10. Thefusion protein of claim 1, wherein the serum half-life of the fusionprotein in the blood or serum of a target animal upon administration islonger than about 3 days.
 11. The fusion protein of claim 1, wherein thetime during which there is a statistically significant decrease in bloodglucose level in a subject relative to a pre-dose level is longer thanone of 2 hours, 6 hours, 9 hours, 12 hours, 18 hours, 1 day, 1.5 days, 2days, 2.5 days, 3 days, 4 days, 5 days, 6 days, 7 days, or longer. 12.The fusion protein of claim 1, wherein the NAOC after the firstsubcutaneous injection in a target animal is greater than 150%FBGL·days·kg/mg.
 13. The fusion protein of claim 12, wherein the ratioof the NAOC after the third weekly subcutaneous injection of the fusionprotein in the target animal to the NAOC after the first subcutaneousinjection of the fusion protein in the target animal is greater than0.50.
 14. The fusion protein of claim 1, wherein the fusion protein isformulated as a pharmaceutical composition.
 15. The pharmaceuticalcomposition of claim 14, wherein the fusion protein is present in thepharmaceutical composition at a concentration of about 3 mg/mL orgreater.
 16. The pharmaceutical composition of claim 14, wherein thecomposition is suitable for subcutaneous administration.
 17. A methodfor lowering the blood glucose level of a target animal, the methodcomprising administering a physiologically effective amount of thefusion protein of claim 1 or a pharmaceutical composition thereof to thetarget animal, and wherein the target animal is a cat.
 18. The method ofclaim 17 in which the target animal is diagnosed with diabetes.
 19. Themethod of claim 17, wherein the fusion protein is administeredsubcutaneously.
 20. The method of claim 17, wherein the fusion proteinis administered daily, twice weekly, or once weekly to the targetanimal.
 21. The method of claim 17, wherein the fusion protein isadministered once weekly to the target animal at a dose between 0.025and 0.5 mg/kg/week.